Antibody drug conjugates (adcs) having enzymatically cleavable groups

ABSTRACT

The invention relates to novel binder-drug conjugates (ADCs) having improved properties, to active metabolites of these ADCs and to processes for their preparation. The present invention furthermore relates to the use of these conjugates for the treatment and/or prevention of diseases and to the use of these conjugates for preparing medicaments for treatment and/or prevention of diseases, in particular hyperproliferative and/or angiogenic disorders such as, for example, cancer diseases.

INTRODUCTION AND STATE OF THE ART

The invention relates to novel binder-drug conjugates (ADCs) havingimproved properties, to active metabolites of these ADCs and toprocesses for their preparation. The present invention furthermorerelates to the use of these conjugates for the treatment and/orprevention of diseases and to the use of these conjugates for preparingmedicaments for treatment and/or prevention of diseases, in particularhyperproliferative and/or angiogenic disorders such as, for example,cancer diseases. Such treatments can be effected as monotherapy or elsein combination with other medicaments or further therapeutic measures.According to the invention, the binder is preferably an antibody.

Cancers are the consequence of uncontrolled cell growth of the mostdiverse tissues. In many cases the new cells penetrate into existingtissue (invasive growth), or they metastasize into remote organs.Cancers occur in a wide variety of different organs and often havetissue-specific courses. The term “cancer” as a generic term thereforedescribes a large group of defined diseases of different organs, tissueand cell types.

Some tumours at early stages can be removed by surgical and radiotherapymeasures. Metastasized tumours as a rule can only be treatedpalliatively by chemotherapeutics. The aim here is to achieve theoptimum combination of an improvement in the quality of life andprolonging of life.

Conjugates of binder proteins with one or more drug molecules are known,in particular in the form of antibody drug conjugates (ADCs) in which aninternalizing antibody directed against a tumour-associated antigen iscovalently attached via a linker to a cytotoxic agent. Followingintroduction of the ADCs into the tumour cell and subsequentdissociation of the conjugate, either the cytotoxic agent itself or acytotoxic metabolite formed therefrom is released within the tumour celland can unfold its action therein directly and selectively. In thismanner, in contrast to conventional cancer chemotherapy, damage tonormal tissue is contained in significantly narrower limits [see, forexample, J. M. Lambert, Curr. Opin. Pharmacol. 5, 543-549 (2005); A. M.Wu and P. D. Senter, Nat. Biotechnol. 23, 1137-1146 (2005); P. D.Senter, Curr. Opin. Chem. Biol. 13, 235-244 (2009); L. Ducry and B.Stump, Bioconjugate Chem. 21, 5-13 (2010)]. Thus, WO2012/171020describes ADCs in which a plurality of toxophore molecules are attachedvia a polymeric linker to an antibody. As possible toxophores,WO2012/171020 mentions, among others, the substances SB 743921, SB715992 (Ispinesib), MK-0371, AZD8477, AZ3146 and ARRY-520.

The substances mentioned last are kinesin spindle protein inhibitors.Kinesin spindle protein (KSP, also known as Eg5, HsEg5, KNSL1 or KIF11)is a kinesin-like motorprotein which is essential for the bipolarmitotic spindle to function. Inhibition of KSP leads to mitotic arrestand, over a relatively long term, to apoptosis (Tao et al., Cancer Cell2005 Jul. 8(1), 39-59). After the discovery of the firstcell-penetrating KSP inhibitor, Monastrol, KSP inhibitors haveestablished themselves as a class of novel chemotherapeutics (Mayer etal., Science 286: 971-974, 1999), and they are subject matter of anumber of patent applications (e.g. WO2006/044825; WO2006/002236;WO2005/051922; WO2006/060737; WO03/060064; WO03/040979; andWO03/049527). However, since KSP is active only during a relativelyshort period of time during the mitosis phase, KSP inhibitors have to bepresent in a sufficiently high concentration during this phase.WO2014/151030 discloses ADCs including certain KSP inhibitors. ADCs withKSP inhibitors which also comprise enzymatically cleavable linkerswhich, however, do not have an optimum activity profile, have alreadybeen disclosed in the patent applications WO2015/096982 andWO2016/096610.

Legumain is a tumour-associated asparaginyl endopeptidase (S. Ishii,Methods Enzymol. 1994, 244, 604; J. M. Chen et al. J. Biol. Chem. 1997,272, 8090) and has been utilized for processing of prodrugs of smallcytotoxic molecules, for example of doxorubicin and etoposidederivatives among others (W. Wu et al. Cancer Res. 2006, 66, 970; L.Stern et al. Bioconjugate Chem. 2009, 20, 500; K.M. Bajjuri et al.ChemMedChem 2011, 6, 54).

Other lysosomal enzymes are, for example, cathepsin or glycosidases, forexample β-glucuronidases, which have also been utilized for release ofthe active compounds by enzymatic cleavage of prodrugs. Groups cleavableenzymatically in vivo are especially 2-8-oligopeptide groups orglycosides. Peptide cleaving sites are disclosed in Bioconjugate Chem.2002, 13, 855-869 and Bioorganic & Medicinal Chemistry Letters 8 (1998)3341-3346 and also Bioconjugate Chem. 1998, 9, 618-626. These include,for example, valine-alanine, valine-lysine, valine-citrulline,alanine-lysine and phenylalanine-lysine (optionally with additionalamide group).

SUMMARY OF THE INVENTION

The prior art discloses various antibody-drug conjugates withenzymatically cleavable linkers which, however, do not have an optimumactivity profile, for example with respect to their broad activity ondifferent cells. It is therefore an object of the present invention toprovide more effective compounds which, after administration at arelatively low concentration, exhibit long-lasting apoptotic action andare therefore of benefit for cancer therapy. Here, on the one hand, theprofile of the metabolites released intracellularly from the ADCs playsan important role. Frequently, the metabolites formed from ADCs aresubstrates of efflux pumps and/or have high cell membrane permeability.Both phenomena may contribute to a short residence time and thussuboptimal apoptotic action in the tumour cell.

Accordingly, the present invention provides binder-drug conjugates(ADCs) having a specific toxophore-linker composition which, inassociation with antibodies, have a particularly interesting activityprofile with respect to potency and activity spectrum. In order tofurther improve the tumour selectivity of ADCs and the metabolitesthereof, binder conjugates have been provided with peptide linkers whichcan be released by lysosomal tumour-associated enzymes such as legumainor cathepsin. The tumour selectivity is thus determined not just by thechoice of antibody but additionally by the enzymatic cleavage of thepeptide derivative, for example by tumour-associated enzymes such aslegumain. Furthermore, the metabolites released from the binder-drugconjugates (ADCs) according to the invention in the tumour cells aredistinguished by a particularly interesting property profile. Theyexhibit low efflux from the tumour cell and lead to high active compoundexposition in tumours. Thus, a high activity is achieved in the tumourcell whereas, owing to poor permeability, there is only low systemiccytotoxic activity, resulting in lower off-target toxicity.

The kinesin spindle protein inhibitors used in accordance with theinvention have an amino group which is essential to the effect. Bymodification of this amino group with peptide derivatives, the effectwith respect to the kinesin spindle protein is blocked and hence thedevelopment of a cytotoxic effect is also inhibited. These peptidederivatives may also be components of the linker to the antibody. Ifthis peptide residue or peptide linker, however, can be released fromthe active compound by tumour-associated enzymes such as legumain orcathepsin, the effect can be re-established in a controlled manner inthe tumour tissue. The particular property profile of the metabolitesformed in the tumour is ensured by a further modification of the kinesinspindle protein inhibitor at a position different from the amino groupin the molecule which, however, does not adversely affect the highpotency at the target.

Furthermore, the structure of the ADCs according to the inventionallows, for certain embodiments, high loading of the antibody (referredto as DAR, drug-to-antibody ratio) which, surprisingly, has no negativeeffect on the physicochemical and pharmakokinetic behaviour of the ADCs.

Surprisingly, it has now been found that binder-drug conjugates of theformula (I)

in which

-   -   X₁ represents N,    -   X₂ represents N and    -   X₃ represents C;        -   or    -   X₁ represents N,    -   X₂ represents C and    -   X₃ represents N;        -   or    -   X₁ represents CH or CF,    -   X₂ represents C and    -   X₃ represents N;        -   or    -   X₁ represents NH,    -   X₂ represents C and    -   X₃ represents C;        -   or    -   X₁ represents CH,    -   X₂ represents N and    -   X₃ represents C,

-   R¹ represents hydrogen or methyl,

-   R² represents methyl, ethyl, —CH₂—CH(CH₃)₂, —CH₂—C(═O)OH or    isopropyl,

-   R³ represents methyl, ethyl, —CH₂—CH(CH₃)₂ or —CH₂—C(═O)—NH₂,

-   M represents the group    -   #-C(═O)—CH(CH₃)—NH—C(═O)—CH₂—NH—C(═O)—CH₂—CH(##)-COOH,    -   #-C(═O)—CH(CH₃)—NH—C(═O)—CH₂—NH—C(═O)—CH(##)-CH₂—COOH,    -   #-C(═O)—CH(CH₃)—NH—C(═O)—CH₂—W,    -   #-C(═O)—CH₂—NH—C(═O)—CH₂—CH(##)-COOH,    -   #-C(═O)—CH₂—NH—C(═O)—CH(##)-CH₂—COOH,    -   #-C(═O)—CH₂—W,    -   #-C(═O)—CH(CH₃)—NH—C(═O)—(CH₂)₂₋₈—C(═O)-###,    -   #-C(═O)—(CH₂)₃—C(═O)-###,    -   #-C(═O)—CH(CH₃)—NH—C(═O)—(CH₂)₅—W,    -   #-C(═O)—CH(CH₃)—NH—C(═O)—(CH₂)-## or    -   #-C(═O)—CH(CH₃)—NH—C(═O)—(CH₂—CH₂—O)₁₋₈—(CH₂)₂—NH—C(═O)—CH₂-##,

-   W represents the group

-   n represents a number from 1 to 50,-   AK represents a binder or a derivative thereof, preferably an    antibody or an antigen-binding fragment,-   # represents the bond to the compound,-   ## represents the bond to a sulfur atom of a cysteine side-chain of    the binder,-   ### represents the bond to a nitrogen atom of a lysine side-chain of    the binder,

and their salts, solvates and salts of these solvates, have superiorproperties compared to the known conjugates.

Preference is given to those binder-drug conjugates of the formula (I),

in which

-   X₁ represents CH,-   X₂ represents C,-   X₃ represents N,-   R¹ represents hydrogen or methyl,-   R² represents methyl, —CH₂—CH(CH₃)₂, —CH₂—C(═O)OH or isopropyl,-   R³ represents methyl, —CH₂—CH(CH₃)₂ or —CH₂—C(═O)—NH₂,-   M represents the group    -   #-C(═O)—CH(CH₃)—NH—C(═O)—CH₂—NH—C(═O)—CH₂—CH(##)-COOH,    -   #-C(═O)—CH(CH₃)—NH—C(═O)—CH₂—NH—C(═O)—CH(##)-CH₂—COOH,    -   #-C(═O)—CH(CH₃)—NH—C(═O)—CH₂—W,    -   #-C(═O)—CH₂—NH—C(═O)—CH₂—CH(##)-COOH,    -   #-C(═O)—CH₂—NH—C(═O)—CH(##)-CH₂—COOH,    -   #-C(═O)—CH₂—W,    -   #-C(═O)—CH(CH₃)—NH—C(═O)—(CH₂)₃—C(═O)-###,    -   #-C(═O)—(CH₂)₃—C(═O)-###,    -   #-C(═O)—CH(CH₃)—NH—C(═O)—(CH₂)₅—W,    -   #-C(═O)—CH(CH₃)—NH—C(═O)—(CH₂)-## or    -   #-C(═O)—CH(CH₃)—NH—C(═O)—(CH₂—CH₂—O)₄—(CH₂)₂—NH—C(═O)—CH₂-##,-   W represents the group

-   n represents a number from 1 to 50,-   AK represents a binder or a derivative thereof, preferably an    antibody or an antigen-binding fragment,-   # represents the bond to the compound,-   ## represents the bond to a sulfur atom of a cysteine side-chain of    the binder,-   ### represents the bond to a nitrogen atom of a lysine side-chain of    the binder,

and their salts, solvates and salts of these solvates.

Particular preference is given to those binder-drug conjugates of theformula (I),

in which

-   R¹ represents hydrogen or methyl,-   R² represents methyl or isopropyl,-   R³ represents methyl or —CH₂—C(═O)—NH₂,-   M represents the group    -   #-C(═O)—CH(CH₃)—NH—C(═O)—(CH₂)₃—C(═O)-###,-   n represents a number from 1 to 50,-   AK represents a binder or a derivative thereof, preferably an    antibody or an antigen-binding fragment,-   # represents the bond to the compound,-   ### represents the bond to a nitrogen atom of a lysine side-chain of    the binder,

and their salts, solvates and salts of these solvates.

Very particular preference is given to those binder-drug conjugates ofthe formula (I), in which

-   R¹ represents methyl,-   R² represents methyl,-   R³ represents —CH₂—C(═O)—NH₂,-   M represents the group    -   #-C(═O)—CH(CH₃)—NH—C(═O)—(CH₂)₃—C(═O)-###,-   n represents a number from 1 to 50,-   AK represents a binder or a derivative thereof, preferably an    antibody or an antigen-binding fragment,-   # represents the bond to the compound,-   ### represents the bond to a nitrogen atom of a lysine side-chain of    the binder,

and their salts, solvates and salts of these solvates.

Special preference is given to those binder-drug conjugates of theformula (I),

in which

-   R¹ represents methyl,-   R² represents methyl,-   R³ represents —CH₂—C(═O)—NH₂,-   M represents the group    -   #-C(═O)—CH(CH₃)—NH—C(═O)—(CH₂)₃—C(═O)-###,-   n represents a number from 1 to 20,-   AK represents a binder or a derivative thereof, preferably an    antibody or an antigen-binding fragment,-   # represents the bond to the compound,-   ### represents the bond to a nitrogen atom of a lysine side-chain of    the binder,

and their salts, solvates and salts of these solvates.

Selected are those binder-drug conjugates of the formula (I),

in which

-   R¹ represents methyl,-   R² represents methyl,-   R³ represents —CH₂—C(═O)—NH₂,-   M represents the group    -   #-C(═O)—CH(CH₃)—NH—C(═O)—(CH₂)₃—C(═O)-###,-   n represents a number from 1 to 20 and-   AK represents an anti-CD123 antibody, an anti-CXCR5 antibody, an    anti-B7H3 antibody, an anti-TWEAKR antibody, an anti-Her2 antibody    or an anti-EGFR antibody or represents an antigen-binding antibody    fragment of these,-   # represents the bond to the compound,-   ### represents the bond to a nitrogen atom of a lysine side-chain of    the antibody (AK) or of the antigen-binding antibody fragment    thereof,

and their salts, solvates and salts of these solvates.

Selected are in particular those binder-drug conjugates of the formula(I),

in which

-   R¹ represents methyl,-   R² represents methyl,-   R³ represents —CH₂—C(═O)—NH₂,-   M represents the group    -   #-C(═O)—CH(CH₃)—NH—C(═O)—(CH₂)₃—C(═O)-###,-   n represents a number from 1 to 20 and-   AK represents an anti-CD123 antibody selected from the group    consisting of TPP-9476, TPP-8988, TPP-8987 and TPP-6013, represents    an anti-CXCR5 antibody selected from the group consisting of    TPP-9574 and TPP-9580, represents an anti-B7H3 antibody TPP-8382,    represents an anti-TWEAKR antibody selected from the group    consisting of TPP-7006 and TPP-7007, represents an anti-Her2    antibody TPP-1015 or represents an anti-EGFR antibody TPP-981 or    represents an antigen-binding antibody fragment of these,-   # represents the bond to the compound,-   ### represents the bond to a nitrogen atom of a lysine side-chain of    the antibody (AK) or of the antigen-binding antibody fragment    thereof,

and their salts, solvates and salts of these solvates.

Preference is given to those binder-drug conjugates of the formulaementioned above in which AK represents a binder which specifically bindsto an extracellular cancer target molecule. In a preferred embodiment,the binder, after binding to its extracellular target molecule on thetarget cell, is internalized by the target cell through the binding.Preferably, the binder is an antibody or an antigen-binding fragment.

In a preferred subject of the invention, the extracellular cancer targetmolecule is selected from the group consisting of the cancer targetmolecules EGFR, CD123, HER2, B7H3, TWEAKR and CXCR5; particularpreference is given to CD123, CXCR5 and B7H3.

In a preferred subject of the invention, the binder AK is an anti-CD123antibody, an anti-CXCR5 antibody, an anti-B7H3 antibody, an anti-TWEAKRantibody, an anti-Her2 antibody or an anti-EGFR antibody, or anantigen-binding antibody fragment of these.

Especially preferred are those binder-drug conjugates of the formulaementioned in which AK (AK1, AK2) represents an antibody selected fromthe group consisting of TPP-8382 (anti B7H3), TPP-6013 (anti-CD123),TPP-8987 (anti-CD123), TPP-8988 (anti-CD123), TPP 9476 (anti-CD123),TPP-9574 (anti-CXCR5) and TPP 9580 (anti-CXCR5), or an antigen-bindingfragment of these. Here, preference is given to the antibodies TPP-6013,TPP-8987, TPP-8988 and TPP-9476 (in each case anti-CD123). The exactstructure (sequence) of these antibodies can be found in the table:Protein sequences of the antibodies, the text following this table andthe sequence listing.

Especially preferred are those binder-drug conjugates of the formula (I)in which AK represents an antibody selected from the group consisting ofTPP-8382 (anti B7H3), TPP-6013 (anti-CD123), TPP-8987 (anti-CD123),TPP-8988 (anti-CD123), TPP 9476 (anti-CD123), TPP-9574 (anti-CXCR5) andTPP 9580 (anti-CXCR5). Here, preference is given to the antibodies (AK)TPP-6013, TPP-8987, TPP-8988 and TPP-9476 (in each case anti-CD123).

DESCRIPTION OF THE FIGURES

FIG. 1: Annotated sequences of preferred antibodies for binder-drugconjugates. What are shown are the protein sequences of the heavy andlight chains of the IgGs, and the VH and VL regions of these antibodies.Below the sequences, important regions are annotated (VH and VL regionsin IgGs, and the CDR regions (H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2,L-CDR3)).

FIG. 2: Sequence listing of sequences of the preferred antibodies forbinder-drug conjugates and of sequences of the target proteins.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides conjugates of a binder or derivative thereof withone or more drug molecules, the drug molecule being a kinesin spindleprotein inhibitor (KSP inhibitor).

Binders which can be used according to the invention, KSP inhibitorsthereof which can be used according to the invention and linkers whichcan be used according to the invention which can be used in combinationwithout any limitation are described below. In particular, the bindersrepresented in each case as preferred or particularly preferred can beemployed in combination with the KSP inhibitors represented in each caseas preferred or particularly preferred, optionally in combination withthe linkers represented in each case as preferred or particularlypreferred.

Particularly Preferred KSP-Inhibitor Conjugates (Binder-Drug Conjugates)

Particular preference is given in accordance with the invention to theKSP inhibitor conjugates which follow, where AK (AK₁, AK₂) representsbinders or a derivative thereof (preferably an antibody), and nrepresents a number from 1 to 20, preferably 1 to 8 and more preferably4 to 8. AK₁ preferably represents an antibody bonded via a cysteineresidue to the KSP inhibitor; AK₂ preferably represents an antibodybonded via a lysine residue to the KSP inhibitor. The binders orantibodies used here are preferably the binders and antibodies describedas preferred in the description.

Here, particular preference is given to the following binder-drugconjugates:

Preference is given to those binder-drug conjugates of the formulaementioned in which AK (AK1, AK2) represents a binder which specificallybinds to an extracellular cancer target molecule. In a preferredembodiment, the binder, after binding to its extracellular targetmolecule on the target cell, is internalized by the target cell throughthe binding.

In a preferred subject of the invention, the extracellular cancer targetmolecule is selected from the group consisting of the cancer targetmolecules EGFR, CD123, Her2, B7H3, TWEAKR and CXCR5, in particularCD123, CXCR5 and B7H3.

In a preferred subject of the invention, the binder AK (AK₁, AK₂) is ananti-CD123 antibody, an anti-CXCR5 antibody, an anti-B7H3 antibody, ananti-TWEAKR antibody, an anti-Her2 antibody or an anti-EGFR antibody, oran antigen-binding antibody fragment of these.

Especially preferred are those binder-drug conjugates of the formulaementioned in which AK (AK1, AK2) represents an antibody selected fromthe group consisting of TPP-8382 (anti B7H3), TPP-6013 (anti-CD123),TPP-8987 (anti-CD123), TPP-8988 (anti-CD123), TPP-9476 (anti-CD123),TPP-9574 (anti-CXCR5) and TPP-9580 (anti-CXCR5), or an antigen-bindingfragment of these. Here, preference is given to the antibodies TPP-6013,TPP-8987, TPP-8988 and TPP-9476 (in each case anti-CD123). The exactstructure (sequence) of these antibodies can be found in the table:Protein sequences of the antibodies, the text following this table andthe sequence listing.

KSP Inhibitor—linker-intermediates and Preparation of the Conjugates

The conjugates according to the invention are prepared by initiallyproviding the low-molecular weight KSP inhibitor thereof with a linker.The intermediate obtained in this manner is then reacted with the binder(preferably antibody).

For an intermediate that couples to a lysine residue and the subsequentcoupling with the antibody, the reaction can be illustrated as follows:

In the above reaction scheme, X₁, X₂, X₃, R¹, R², R³ and AK₂ have themeanings given in the formula (I) and here R⁴ represents methyl and nrepresents 0 or 1.

The synthesis of building block A has been described in WO2015/096982.The peptide derivatives B and C were prepared by classical methods ofpeptide chemistry. Intermediates C and D were coupled using HATU in DMFin the presence of N,N-diisopropylethylamine at RT. Subsequently, boththe benzyloxycarbonyl protective group and the benzyl ester were removedhydrogenolytically over 10% palladium on activated carbon. Thecompletely deprotected intermediate was then reacted with1,1′-[(1,5-dioxopentan-1,5-diyl)bis(oxy)]dipyrrolidine-2,5-dione in DMFin the presence of N,N-diisopropylethylamine at RT to give the ADCprecursor molecule E. This activated ester was then coupled with therespective antibodies as described in Chapter B-5.

For an intermediate that couples to a cysteine residue and thesubsequent coupling with the antibody, the reaction can be illustratedas follows:

In the above reaction scheme, X₁, X₂, X₃, R¹, R², R³ and AK₁ have themeanings given in the formula (I) and here R⁴ represents methyl and nrepresents 1.

Using an analogous procedure, it is also possible to prepare compoundsin which n represents 0.

The synthesis of building block A has been described in WO2015/096982.The peptide derivatives B and C were prepared by classical methods ofpeptide chemistry. Intermediates C and D were coupled using HATU in DMFin the presence of N,N-diisopropylethylamine at RT. Subsequently, boththe benzyloxycarbonyl protective group and the benzyl ester were removedhydrogenolytically over 10% palladium on activated carbon. Thecompletely deprotected intermediate was then reacted with1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-1H-pyrrole-2,5-dione inDMF in the presence of N,N-diisopropylethylamine at RT to give the ADCprecursor molecule E. This maleimide derivative was then coupled withthe respective antibodies as described in Chapter B-4.

Depending on the linker, succinimide-linked ADCs may, after conjugation,be converted into the open-chain succinamides, which have anadvantageous stability profile.

This reaction (ring opening) can be effected at pH 7.5 to 9, preferablyat pH 8, at a temperature of 25° C. to 37° C., for example by stirring.The preferred stirring time is 8 to 30 hours.

For an intermediate that couples to a cysteine residue and thesubsequent coupling with the antibody followed by ring opening of thesuccinimide ring, the reaction can be illustrated as follows:

In the above reaction scheme, X₁, X₂, X₃, R¹, R², R³ and AK₁ have themeanings given in the formula (I) and here R⁴ represents methyl and nrepresents 0 or 1.

The synthesis of building block A has been described in WO2015/096982.The peptide derivatives B and C were prepared by classical methods ofpeptide chemistry. Intermediates C and D were coupled using HATU in DMFin the presence of N,N-diisopropylethylamine at RT. Subsequently, boththe benzyloxycarbonyl protective group and the benzyl ester were removedhydrogenolytically over 10% palladium on activated carbon. Thecompletely deprotected intermediate was then reacted with1-{2-[(2,5-dioxopyrrolidin-1-yl)oxy]-2-oxoethyl}-1H-pyrrole-2,5-dione inthe presence of N,N-diisopropylethylamine at RT to give the ADCprecursor molecule E. This maleimide derivative was then coupled withthe respective antibodies as described in Chapter B-4 under small scalecoupling or under medium scale coupling.

Binders

In the broadest sense, the term “binder” is understood to mean amolecule which binds to a target molecule present at a certain targetcell population to be addressed by the binder-drug conjugate. The termbinder is to be understood in its broadest meaning and also comprises,for example, lectins, proteins capable of binding to certain sugarchains, or phospholipid-binding proteins. Such binders include, forexample, high-molecular weight proteins (binding proteins), polypeptidesor peptides (binding peptides), non-peptidic (e.g. aptamers (U.S. Pat.No. 5,270,163) review by Keefe A D., et al., Nat. Rev. Drug Discov.2010; 9:537-550), or vitamins) and all other cell-binding molecules orsubstances. Binding proteins are, for example, antibodies and antibodyfragments or antibody mimetics, for example affibodies, adnectins,anticalins, DARPins, avimers, nanobodies (review by Gebauer M. et al.,Curr. Opinion in Chem. Biol. 2009; 13:245-255; Nuttall S. D. et al.,Curr. Opinion in Pharmacology 2008; 8:608-617). Binding peptides are,for example, ligands of a ligand/receptor pair such as, for example,VEGF of the ligand/receptor pair VEGF/KDR, such as transferrin of theligand/receptor pair transferrin/transferrin receptor orcytokine/cytokine receptor, such as TNFalpha of the ligand/receptor pairTNFalpha/TNFalpha receptor.

The binder may be a binding protein. Preferred embodiments of thebinders are an antibody, an antigen-binding antibody fragment, amultispecific antibody or an antibody mimetic.

The literature also discloses various options of covalent coupling(conjugation) of organic molecules to binders and in particularantibodies. Preference according to the invention is given to theconjugation of the toxophores to the antibody via one or more sulfuratoms of cysteine residues of the antibody and/or via one or more NHgroups of lysine residues of the antibody. However, it is also possibleto bind the toxophore to the antibody via free carboxyl groups or viasugar residues of the antibody.

A “target molecule” in the broadest sense is understood to mean amolecule which is present in the target cell population and which may bea protein (for example a receptor of a growth factor) or a non-peptidicmolecule (for example a sugar or phospholipid). It is preferably areceptor or an antigen.

The term “extracellular” target molecule describes a target molecule,attached to the cell, which is located at the outside of a cell, or thepart of a target molecule which is located at the outside of a cell,i.e. a binder may bind on an intact cell to its extracellular targetmolecule. An extracellular target molecule may be anchored in the cellmembrane or be a component of the cell membrane. The person skilled inthe art is aware of methods for identifying extracellular targetmolecules. For proteins, this may be by determining the transmembranedomain(s) and the orientation of the protein in the membrane. These dataare usually deposited in protein databases (e.g. SwissProt).

The term “cancer target molecule” describes a target molecule which ismore abundantly present on one or more cancer cell species than onnon-cancer cells of the same tissue type. Preferably, the cancer targetmolecule is selectively present on one or more cancer cell speciescompared with non-cancer cells of the same tissue type, whereselectively describes an at least two-fold enrichment on cancer cellscompared to non-cancer cells of the same tissue type (a “selectivecancer target molecule”). The use of cancer target molecules allows theselective therapy of cancer cells using the conjugates according to theinvention.

The binder can be attached to the linker via a bond. The binder can belinked by means of a heteroatom of the binder. Heteroatoms according tothe invention of the binder which can be used for attachment are sulfur(in one embodiment via a sulfhydryl group of the binder), oxygen(according to the invention by means of a carboxyl or hydroxyl group ofthe binder) and nitrogen (in one embodiment via a primary or secondaryamine group or amide group of the binder). These heteroatoms may bepresent in the natural binder or are introduced by chemical methods ormethods of molecular biology. According to the invention, the attachmentof the binder to the toxophore has only a minor effect on the bindingactivity of the binder with respect to the target molecule. In apreferred embodiment, the linkage has no effect on the binding activityof the binder with respect to the target molecule.

In accordance with the present invention, the term “antibody” is to beunderstood in its broadest meaning and comprises immunoglobulinmolecules, for example intact or modified monoclonal antibodies,polyclonal antibodies or multispecific antibodies (e.g. bispecificantibodies). An immunoglobulin molecule preferably comprises a moleculehaving four polypeptide chains, two heavy chains (H chains) and twolight chains (L chains) which are typically linked by disulfide bridges.Each heavy chain comprises a variable domain of the heavy chain(abbreviated VH) and a constant domain of the heavy chain. The constantdomain of the heavy chain may, for example, comprise three domains CH1,CH2 and CH3. Each light chain comprises a variable domain (abbreviatedVL) and a constant domain. The constant domain of the light chaincomprises a domain (abbreviated CL). The VH and VL domains may besubdivided further into regions having hypervariability, also referredto as complementarity determining regions (abbreviated CDR) and regionshaving low sequence variability (framework region, abbreviated FR).Typically, each VH and VL region is composed of three CDRs and up tofour FRs. For example from the amino terminus to the carboxy terminus inthe following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. An antibodymay be obtained from any suitable species, e.g. rabbit, llama, camel,mouse or rat. In one embodiment, the antibody is of human or murineorigin. An antibody may, for example, be human, humanized or chimeric.

The term “monoclonal” antibody refers to antibodies obtained from apopulation of substantially homogeneous antibodies, i.e. individualantibodies of the population are identical except for naturallyoccurring mutations, of which there may be a small number. Monoclonalantibodies recognize a single antigenic binding site with highspecificity. The term monoclonal antibody does not refer to a particularpreparation process.

The term “intact” antibody refers to antibodies comprising both anantigen-binding domain and the constant domain of the light and heavychain. The constant domain may be a naturally occurring domain or avariant thereof having a number of modified amino acid positions, andmay also be aglycosylated.

The term “modified intact” antibody refers to intact antibodies fusedvia their amino terminus or carboxy terminus by means of a covalent bond(e.g. a peptide bond) with a further polypeptide or protein notoriginating from an antibody. Furthermore, antibodies may be modifiedsuch that, at defined positions, reactive cysteines are introduced tofacilitate coupling to a toxophore (see Junutula et al. Nat Biotechnol.2008 August; 26(8):925-32).

“Amino acid modification” or “mutation” here means an amino acidsubstitution, insertion and/or deletion in a polypeptide sequence. Thepreferred amino acid modification here is a substitution. “Amino acidsubstitution” or “substitution” here means an exchange of an amino acidat a given position in a protein sequence for another amino acid. Forexample, the substitution Y50W describes a variant of a parentpolypeptide in which the tyrosine at position 50 has been exchanged fora tryptophan. A “variant” of a polypeptide describes a polypeptidehaving an amino acid sequence substantially identical to a referencepolypeptide, typically a native or “parent” polypeptide. The polypeptidevariant may have one or more amino acid exchanges, deletions and/orinsertions at particular positions in the native amino acid sequence.

The term “human” antibody refers to antibodies which can be obtainedfrom a human or which are synthetic human antibodies. A “synthetic”human antibody is an antibody which is partially or entirely obtainablein silico from synthetic sequences based on the analysis of humanantibody sequences. A human antibody can be encoded, for example, by anucleic acid isolated from a library of antibody sequences of humanorigin. An example of such an antibody can be found in Soderlind et al.,Nature Biotech. 2000, 18:853-856. Such “human” and “synthetic”antibodies also include aglycosylated variants which have been producedeither by deglycosylation by PNGaseF or by mutation of N297 (Kabatnumbering) of the heavy chain to any other amino acid.

The term “humanized” or “chimeric” antibody describes antibodiesconsisting of a non-human and a human portion of the sequence. In theseantibodies, part of the sequences of the human immunoglobulin(recipient) is replaced by sequence portions of a non-humanimmunoglobulin (donor). In many cases, the donor is a murineimmunoglobulin. In the case of humanized antibodies, amino acids of theCDR of the recipient are replaced by amino acids of the donor.Sometimes, amino acids of the framework, too, are replaced bycorresponding amino acids of the donor. In some cases the humanizedantibody contains amino acids present neither in the recepient nor inthe donor, which were introduced during the optimization of theantibody. In the case of chimeric antibodies, the variable domains ofthe donor immunoglobulin are fused with the constant regions of a humanantibody. Such “humanized” and “chimeric” antibodies also includeaglycosylated variants which have been produced either bydeglycosylation by PNGaseF or by mutation of N297 (Kabat numbering) ofthe heavy chain to any other amino acid.

The term complementarity determining region (CDR) as used herein refersto those amino acids of a variable antibody domain which are requiredfor binding to the antigen. Typically, each variable region has threeCDR regions referred to as CDR1, CDR2 and CDR3. Each CDR region mayembrace amino acids according to the definition of Kabat and/or aminoacids of a hypervariable loop defined according to Chotia. Thedefinition according to Kabat comprises, for example, the region fromabout amino acid position 24-34 (CDR1), 50-56 (CDR2) and 89-97 (CDR3) ofthe variable light chain/domain (VL) and 31-35 (CDR1), 50-65 (CDR2) and95-102 (CDR3) of the variable heavy chain/domain (VH) (Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991)). Thedefinition according to Chotia comprises, for example, the region fromabout amino acid position 26-32 (CDR1), 50-52 (CDR2) and 91-96 (CDR3) ofthe variable light chain (VL) and 26-32 (CDR1), 53-55 (CDR2) and 96-101(CDR3) of the variable heavy chain (VH) (Chothia and Lesk; J Mol Biol196: 901-917 (1987)). In some cases, a CDR may comprise amino acids froma CDR region defined according to Kabat and Chotia.

Depending on the amino acid sequence of the constant domain of the heavychain, antibodies may be categorized into different classes. There arefive main classes of intact antibodies: IgA, IgD, IgE, IgG and IgM, andseveral of these can be divided into further subclasses. (Isotypes),e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The constant domains of theheavy chain, which correspond to the different classes, are referred toas [alpha/α], [delta/δ], [epsilon/ε], [gamma/γ] and [my/μ]. Both thethree-dimensional structure and the subunit structure of antibodies areknown.

The term “functional fragment” or “antigen-binding antibody fragment” ofan antibody/immunoglobulin is defined as a fragment of anantibody/immunoglobulin (e.g. the variable domains of an IgG) whichstill comprise the antigen binding domains of theantibody/immunoglobulin. The “antigen binding domain” of an antibodytypically comprises one or more hypervariable regions of an antibody,for example the CDR, CDR2 and/or CDR3 region. However, the “framework”or “skeleton” region of an antibody may also play a role during bindingof the antibody to the antigen. The framework region forms the skeletonof the CDRs. Preferably, the antigen binding domain comprises at leastamino acids 4 to 103 of the variable light chain and amino acids 5 to109 of the variable heavy chain, more preferably amino acids 3 to 107 ofthe variable light chain and 4 to 111 of the variable heavy chain,especially preferably the complete variable light and heavy chains, i.e.amino acids 1-109 of the VL and 1 to 113 of the VH (numbering accordingto WO97/08320).

“Functional fragments” or “antigen-binding antibody fragments” of theinvention encompass, non-conclusively, Fab, Fab′, F(ab′)₂ and Fvfragments, diabodies, Single Domain Antibodies (DAbs), linearantibodies, individual chains of antibodies (single-chain Fv,abbreviated to scFv); and multispecific antibodies, such as bi- andtri-specific antibodies, for example, formed from antibody fragments C.A. K Borrebaeck, editor (1995) Antibody Engineering (Breakthroughs inMolecular Biology), Oxford University Press; R. Kontermann & S. Duebel,editors (2001) Antibody Engineering (Springer Laboratory Manual),Springer Verlag. Antibodies other than “multispecific” or“multifunctional” antibodies are those having identical binding sites.Multispecific antibodies may be specific for different epitopes of anantigen or may be specific for epitopes of more than one antigen (see,for example, WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt,et al., 1991, J. Immunol. 14760 69; U.S. Pat. Nos. 4,474,893; 4,714,681;4,925,648; 5,573,920; 5,601,819; or Kostelny et al., 1992, J. Immunol.148 1547 1553). An F(ab′)₂ or Fab molecule may be constructed such thatthe number of intermolecular disulfide interactions occurring betweenthe Ch1 and the CL domains can be reduced or else completely prevented.

“Epitopes” refer to protein determinants capable of binding specificallyto an immunoglobulin or T cell receptors. Epitopic determinants usuallyconsist of chemically active surface groups of molecules such as aminoacids or sugar side chains or combinations thereof, and usually havespecific 3-dimensional structural properties and also specific chargeproperties.

“Functional fragments” or “antigen-binding antibody fragments” may befused with another polypeptide or protein, not originating from anantibody, via the amino terminus or carboxyl terminus thereof, by meansof a covalent bond (e.g. a peptide linkage). Furthermore, antibodies andantigen-binding fragments may be modified by introducing reactivecysteines at defined locations, in order to facilitate coupling to atoxophore (see Junutula et al. Nat Biotechnol. 2008 August;26(8):925-32).

Polyclonal antibodies can be prepared by methods known to a person ofordinary skill in the art. Monoclonal antibodies may be prepared bymethods known to a person of ordinary skill in the art (Köhler andMilstein, Nature, 256, 495-497, 1975). Human and humanized monoclonalantibodies may be prepared by methods known to a person of ordinaryskill in the art (Olsson et al., Meth Enzymol. 92, 3-16 or Cabilly et alU.S. Pat. No. 4,816,567 or Boss et al U.S. Pat. No. 4,816,397).

A person of ordinary skill in the art is aware of diverse methods forpreparing human antibodies and fragments thereof, such as, for example,by means of transgenic mice (N Lonberg and D Huszar, Int Rev Immunol.1995; 13(1):65-93) or Phage Display Technologien (Clackson et al.,Nature. 1991 Aug 15;352(6336):624-8). Antibodies of the invention may beobtained from recombinant antibody libraries consisting for example onthe amino acid sequences of a multiplicity of antibodies compiled from alarge number of healthy volunteers. Antibodies may also be produced bymeans of known recombinant DNA technologies. The nucleic acid sequenceof an antibody can be obtained by routine sequencing or is availablefrom publically accessible databases.

An “isolated” antibody or binder has been purified to remove otherconstituents of the cell. Contaminating constituents of a cell which mayinterfere with a diagnostic or therapeutic use are, for example,enzymes, hormones, or other peptidic or non-peptidic constituents of acell. A preferred antibody or binder is one which has been purified toan extent of more than 95% by weight, relative to the antibody or binder(determined for example by Lowry method, UV-Vis spectroscopy or by SDScapillary gel electrophoresis). Moreover an antibody which has beenpurified to such an extent that it is possible to determine at least 15amino acids of the amino terminus or of an internal amino acid sequence,or which has been purified to homogeneity, the homogeneity beingdetermined by SDS-PAGE under reducing or non-reducing conditions(detection may be determined by means of Coomassie Blue staining orpreferably by silver coloration). However, an antibody is normallyprepared by one or more purification steps.

The term “specific binding” or “binds specifically” refers to anantibody or binder which binds to a predetermined antigen/targetmolecule. Specific binding of an antibody or binder typically describesan antibody or binder having an affinity of at least 10⁻⁷ M (as Kdvalue; i.e. preferably those with Kd values smaller than 10⁻⁷ M), withthe antibody or binder having an at least two times higher affinity forthe predetermined antigen/target molecule than for a non-specificantigen/target molecule (e.g. bovine serum albumin, or casein) which isnot the predetermined antigen/target molecule or a closely relatedantigen/target molecule. Specific binding of an antibody or binder doesnot exclude the antibody or binder binding to a plurality ofantigens/target molecules (e.g. orthologs of different species). Theantibodies preferably have an affinity of at least 10⁻⁷ M (as Kd value;in other words preferably those with smaller Kd values than 10⁻⁷ M),preferably of at least 10⁻⁹ M, more preferably in the range from 10⁻⁹ Mto 10⁻¹¹ M. The Kd values may be determined, for example, by means ofsurface plasmon resonance spectroscopy.

The antibody-drug conjugates of the invention likewise exhibitaffinities in these ranges. The affinity is preferably not substantiallyaffected by the conjugation of the drugs (in general, the affinity isreduced by less than one order of magnitude, in other words, forexample, at most from 10⁻⁹ M to 10⁻⁷ M).

The antibodies used in accordance with the invention are also notablepreferably for a high selectivity. A high selectivity exists when theantibody of the invention exhibits an affinity for the target proteinwhich is better by a factor of at least 2, preferably by a factor of 5or more preferably by a factor of 10, than for an independent otherantigen, e.g. human serum albumin (the affinity may be determined, forexample, by means of surface plasmon resonance spectroscopy).

Furthermore, the antibodies of the invention that are used arepreferably cross-reactive. In order to be able to facilitate and betterinterpret preclinical studies, for example toxicological or activitystudies (e.g. in xenograft mice), it is advantageous if the antibodyused in accordance with the invention not only binds the human targetprotein but also binds the species target protein in the species usedfor the studies. In one embodiment the antibody used in accordance withthe invention, in addition to the human target protein, iscross-reactive to the target protein of at least one further species.For toxicological and activity studies it is preferred to use species ofthe families of rodents, dogs and non-human primates. Preferred rodentspecies are mouse and rat. Preferred non-human primates are rhesusmonkeys, chimpanzees and long-tailed macaques.

In one embodiment the antibody used in accordance with the invention, inaddition to the human target protein, is cross-reactive to the targetprotein of at least one further species selected from the group ofspecies consisting of mouse, rat and long-tailed macaque (Macacafascicularis). Especially preferred are antibodies used in accordancewith the invention which in addition to the human target protein are atleast cross-reactive to the mouse target protein. Preference is given tocross-reactive antibodies whose affinity for the target protein of thefurther non-human species differs by a factor of not more than 50, moreparticularly by a factor of not more than ten, from the affinity for thehuman target protein.

Antibodies Directed Against a Cancer Target Molecule

The target molecule towards which the binder, for example an antibody oran antigen-binding fragment thereof, is directed is preferably a cancertarget molecule. The term “cancer target molecule” describes a targetmolecule which is more abundantly present on one or more cancer cellspecies than on non-cancer cells of the same tissue type. Preferably,the cancer target molecule is selectively present on one or more cancercell species compared with non-cancer cells of the same tissue type,where selectively describes an at least two-fold enrichment on cancercells compared to non-cancer cells of the same tissue type (a “selectivecancer target molecule”). The use of cancer target molecules allows theselective therapy of cancer cells using the conjugates according to theinvention.

Antibodies which are specific against an antigen, for example cancercell antigen, can be prepared by a person of ordinary skill in the artby means of methods with which he or she is familiar (such asrecombinant expression, for example) or may be acquired commercially (asfor example from Merck KGaA, Germany). Examples of known commerciallyavailable antibodies in cancer therapy are Erbitux® (cetuximab, MerckKGaA), Avastin® (bevacizumab, Roche) and Herceptin® (trastuzumab,Genentech). Trastuzumab is a recombinant humanized monoclonal antibodyof the IgG1kappa type which in a cell-based assay (Kd=5 nM) binds theextracellular domains of the human epidermal growth receptor with highaffinity. The antibody is produced recombinantly in CHO cells. All theseantibodies can also be produced as aglycosylated variants of theseantibodies, either by deglycosylation by means of PNGase F or bymutation of N297 (Kabat numbering) of the heavy chain to any amino acid.

In a preferred embodiment, the target molecule is a selective cancertarget molecule.

In a particularly preferred embodiment, the target molecule is aprotein.

In one embodiment, the target molecule is an extracellular targetmolecule. In a preferred embodiment, the extracellular target moleculeis a protein.

Cancer target molecules are known to those skilled in the art. Examplesof these are listed below.

Examples of cancer target molecules are:

(1) EGFR (EGF receptor, NCBI Reference Sequence NP_005219.2, NCBI GeneID: 1956)

(2) mesothelin (SwissProt Reference Q13421-3), mesothelin being encodedby amino acids 296-598. Amino acids 37-286 code formegakaryocyte-potentiating factor. Mesothelin is anchored in the cellmembrane by a GPI anchor and is localized extracellularly.

(3) Carboanhydrase IX (CA9, SwissProt Reference Q16790), NCBI Gene ID:768)

(4) C4.4a (NCBI Reference Sequence NP_055215.2; synonym LYPD3, NCBI GeneID: 27076)

(5) CD52 (NCBI Reference Sequence NP_001794.2)

(6) Her2 (ERBB2; NCBI Reference Sequence NP_004439.2; NCBI Gene ID:2064)

(7) CD20 (NCBI Reference Sequence NP_068769.2)

(8) the lymphocyte activation antigen CD30 (SwissProt ID P28908)

(9) the lymphocyte adhesion molecule CD22 (SwissProt ID P20273; NCBIGene ID: 933)

(10) the myloid cell surface antigen CD33 (SwissProt ID P20138; NCBIGene ID: 945)

(11) the transmembrane glycoprotein NMB (GPNMB, SwissProt ID Q14956,NCBI Gene ID: 10457)

(12) the adhesion molecule CD56 (SwissProt ID P13591)

(13) the surface molecule CD70 (SwissProt ID P32970, NCBI Gene ID: 970)

(14) the surface molecule CD74 (SwissProt ID P04233, NCBI Gene ID: 972)

(15) the B-lymphocyte antigen CD19 (SwissProt ID P15391, NCBI Gene ID:930)

(16) the surface protein Mucin-1 (MUC1, SwissProt ID P15941, NCBI GeneID: 4582)

(17) the surface protein CD138 (SwissProt ID P18827)

(18) the integrin alphaV (NCBI reference sequence: NP_002201.1, NCBIGene ID: 3685)

(19) the teratocarcinoma-derived growth factor 1 protein TDGF1 (NCBIReference Sequence: NP_003203.1, NCBI Gene ID: 6997)

(20) the prostate-specific membrane antigen PSMA (Swiss Prot ID: Q04609;NCBI Gene ID: 2346)

(21) the tyrosine protein kinase EPHA2 (Swiss Prot ID: P29317, NCBI GeneID: 1969)

(22) the surface protein SLC44A4 (NCBI Reference Sequence:NP_001171515.1, NCBI Gene ID: 80736)

(23) the surface protein BMPR1B (SwissProt: 000238)

(24) the transport protein SLC7A5 (SwissProt: 001650)

(25) the epithelial prostate antigen STEAP1 (SwissProt: Q9UHE8, Gene ID:26872)

(26) the ovarian carcinoma antigen MUC16 (SwissProt: Q8WXI7, Gene ID:94025)

(27) the transport protein SLC34A2 (SwissProt: 095436, Gene ID: 10568)

(28) the surface protein SEMA5b (SwissProt: Q9P283)

(29) the surface protein LYPD1 (SwissProt: Q8N2G4)

(30) the endothelin receptor type B EDNRB (SwissProt: P24530, NCBI GeneID: 1910)

(31) the ring finger protein RNF43 (SwissProt: Q68DV7)

(32) the prostate carcinoma-associated protein STEAP2 (SwissProt:Q8NFT2)

(33) the cation channel TRPM4 (SwissProt: Q8TD43)

(34) the complement receptor CD21 (SwissProt: P20023)

(35) the B-cell antigen receptor complex-associated protein CD79b(SwissProt: P40259, NCBI Gene ID: 974)

(36) the cell adhesion antigen CEACAM6 (SwissProt: P40199)

(37) the dipeptidase DPEP1 (SwissProt: P16444)

(38) the interleukin receptor IL20Ralpha (SwissProt: Q9UHF4, NCBI GeneID: 3559)

(39) the proteoglycan BCAN (SwissProt: Q96GW7)

(40) the ephrin receptor EPHB2 (SwissProt: P29323)

(41) the prostate stem cell-associated protein PSCA (NCBI ReferenceSequence: NP_005663.2)

(42) the surface protein LHFPL3 (SwissProt: Q86UP9)

(43) the receptor protein TNFRSF13C (SwissProt: Q96RJ3)

(44) the B-cell antigen receptor complex-associated protein CD79a(SwissProt: P11912)

(45) the receptor protein CXCR5 (CD185; SwissProt: P32302; NCBI Gene ID643, NCBI Reference Sequence: NP_001707.1)

(46) the ion channel P2X5 (SwissProt: Q93086)

(47) the lymphocyte antigen CD180 (SwissProt: Q99467)

(48) the receptor protein FCRL1 (SwissProt: Q96LA6)

(49) the receptor protein FCRL5 (SwissProt: Q96RD9)

(50) the MHC class II molecule Ia antigen HLA-DOB (NCBI ReferenceSequence: NP_002111.1)

(51) the T-cell protein VTCN1 (SwissProt: Q7Z7D3)

(52) TWEAKR (FN14, TNFRSF12A, NCBI Reference Sequence: NP_057723.1, NCBIGene ID: 51330)

(53) the lymphocyte antigen CD37 (Swiss Prot: P11049, NCBI Gene ID: 951)

(54) the FGF receptor 2; FGFR2 (NCBI Gene ID: 2263; Official Symbol:FGFR2). FGFR2 receptor occurs in different splice variants (alpha, beta,IIIb, IIIc). All splice variants can act as target molecule.

(55) the transmembrane glycoprotein B7H3 (CD276; NCBI Gene ID: 80381NCBI Reference Sequence: NP_001019907.1, Swiss Prot: Q5ZPR3-1)

(56) the B cell receptor BAFFR (CD268; NCBI Gene ID:115650)

(57) the receptor protein ROR 1 (NCBI Gene ID: 4919)

(58) the surface receptor CD123 (IL3RA; NCBI Gene ID: 3563; NCBIReference Sequence: NP_002174.1; Swiss-Prot: P26951)

(59) the receptor protein syncytin (NCBI Gene ID 30816)

(60) aspartate beta-hydroxylase (ASPH; NCBI Gene ID 444)

(61) the cell surface glycoprotein CD44 (NCBI Gene ID: 960)

(62) CDH15 (Cadherin 15, NCBI Gene ID: 1013)

(63) the cell surface glycoprotein CEACAMS (NCBI Gene ID: 1048)

(64) the cell adhesion molecule L1-like (CHL1, NCBI Gene ID: 10752)

(65) the receptor tyrosine kinase c-Met (NCBI Gene ID: 4233)

(66) the notch ligand DLL3 (NCBI Gene ID: 10683)

(67) the ephrin A4 (EFNA4, NCBI Gene ID: 1945)

(68) ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3, NCBIGene ID: 5169)

(69) coagulation factor III (F3, NCBI Gene ID: 2152)

(70) FGF receptor 3 (FGFR3, NCBI Gene ID: 2261)

(71) the folate hydrolase FOLH1 (NCBI Gene ID: 2346)

(72) the folate receptor 1 (FOLR1; NCBI Gene ID: 2348)

(73) the guanylate cyclase 2C (GUCY2C, NCBI Gene ID: 2984)

(74) the KIT proto-oncogen receptor tyrosine kinase (NCBI Gene ID: 3815)

(75) lysosomal-associated membrane protein 1 (LAMP1, NCBI Gene ID: 3916)

(76) lymphocyte antigen 6 complex, locus E (LY6E, NCBI Gene ID: 4061)

(77) the protein NOTCH3 (NCBI Gene ID: 4854)

(78) protein tyrosine kinase 7 (PTK7, NCBI Gene ID: 5754)

(79) nectin cell adhesion molecule 4 (PVRL4, NECTIN4, NCBI Gene ID:81607)

(80) the transmembrane protein syndecan 1 (SDC1, NCBI Gene ID: 6382)

(81) SLAM family member 7 (SLAMF7, NCBI Gene ID: 57823)

(82) the transport protein SLC39A6 (NCBI Gene ID: 25800)

(83) SLIT- and NTRK-like family member 6 (SLITRK6, NCBI Gene ID: 84189)

(84) the cell surface receptor TACSTD2 (NCBI Gene ID: 4070)

(85) the receptor protein TNFRSF8 (NCBI Gene ID: 943)

(86) the receptor protein TNFSF13B (NCBI Gene ID: 10673)

(87) the glycoprotein TPBG (NCBI Gene ID: 7162)

(88) the cell surface receptor TROP2 (TACSTD2, NCBI Gene ID: 4070)

(89) the galanin-like G protein-coupled receptor KISS1R (GPR54, NCBIGene ID: 84634)

(90) the transport protein SLAMF6 (NCBI Gene ID: 114836)

In a preferred subject of the invention, the cancer target molecule isselected from the group consisting of the cancer target molecules EGFR,CD123, Her2, B7H3, TWEAKR and CXCR5, in particular CD123, CXCR5 andB7H3.

In a further particularly preferred subject of the invention, the binderbinds to an extracellular cancer target molecule selected from the groupconsisting of the cancer target molecules EGFR, CD123, Her2, B7H3,TWEAKR and CXCR5, in particular CD123, CXCR5 and B7H3.

In a further particularly preferred subject of the invention, the binderbinds specifically to an extracellular cancer target molecule selectedfrom the group consisting of the cancer target molecules EGFR, CD123,Her2, B7H3, TWEAKR and CXCR5, in particular CD123, CXCR5 and B7H3. In apreferred embodiment, the binder, after binding to its extracellulartarget molecule on the target cell, is internalized by the target cellthrough the binding. This causes the binder-drug conjugate, which may bean immunoconjugate or an ADC, to be taken up by the target cell. Thebinder is then processed, preferably intracellularly, with preferencelysosomally.

In one embodiment the binder is a binding protein. In a preferredembodiment the binder is an antibody, an antigen-binding antibodyfragment, a multispecific antibody or an antibody mimetic.

Preferred antibody mimetics are affibodies, adnectins, anticalins,DARPins, avimers, or nanobodies. Preferred multispecific antibodies arebispecific and trispecific antibodies.

In a preferred embodiment the binder is an antibody or anantigen-binding antibody fragment, more preferably an isolated antibodyor an isolated antigen-binding antibody fragment.

Preferred antigen-binding antibody fragments are Fab, Fab′, F(ab′)2 andFv fragments, diabodies, DAbs, linear antibodies and scFv. Particularlypreferred are Fab, diabodies and scFv.

In a particularly preferred embodiment the binder is an antibody.Particularly preferred are monoclonal antibodies or antigen-bindingantibody fragments thereof. Further particularly preferred are human,humanized or chimeric antibodies or antigen-binding antibody fragmentsthereof.

Antibodies or antigen-binding antibody fragments which bind cancertarget molecules may be prepared by a person of ordinary skill in theart using known processes, such as, for example, chemical synthesis orrecombinant expression. Binders for cancer target molecules may beacquired commercially or may be prepared by a person of ordinary skillin the art using known processes, such as, for example, chemicalsynthesis or recombinant expression. Further processes for preparingantibodies or antigen-binding antibody fragments are described in WO2007/070538 (see page 22 “Antibodies”). The person skilled in the artknows how processes such as phage display libraries (e.g. MorphosysHuCAL Gold) can be compiled and used for discovering antibodies orantigen-binding antibody fragments (see WO 2007/070538, page 24 ff andAK Example 1 on page 70, AK Example 2 on page 72). Further processes forpreparing antibodies that use DNA libraries from B cells are describedfor example on page 26 (WO 2007/070538). Processes for humanizingantibodies are described on page 30-32 of WO2007070538 and in detail inQueen, et al., Pros. Natl. Acad. Sci. USA 8610029-10033,1989 or in WO90/0786. Furthermore, processes for recombinant expression of proteinsin general and of antibodies in particular are known to the personskilled in the art (see, for example, in Berger and Kimrnel (Guide toMolecular Cloning Techniques, Methods in Enzymology, Vol. 152, AcademicPress, Inc.); Sambrook, et al., (Molecular Cloning A Laboratory Manual,(Second Edition, Cold Spring Harbor Laboratory Press; Cold SpringHarbor, N.Y.; 1989) Vol. 1-3); Current Protocols in Molecular Biology,(F. M. Ausabel et al. [Eds.], Current Protocols, Green PublishingAssociates, Inc./John Wiley & Sons, Inc.); Harlow et al., (MonoclonalAntibodies A Laboratory Manual, Cold Spring Harbor Laboratory Press(19881, Paul [Ed.]); Fundamental Immunology, (Lippincott Williams &Wilkins (1998)); and Harlow, et al., (Using Antibodies A LaboratoryManual, Cold Spring Harbor Laboratory Press (1998)). The person skilledin the art knows the corresponding vectors, promoters and signalpeptides which are necessary for the expression of a protein/antibody.Commonplace processes are also described in WO 2007/070538 on pages41-45. Processes for preparing an IgG1 antibody are described forexample in WO 2007/070538 in Example 6 on page 74 ff. Processes whichallow the determination of the internalization of an antibody afterbinding to its antigen are known to the skilled person and are describedfor example in WO 2007/070538 on page 80. The person skilled in the artis able to use the processes described in WO 2007/070538 that have beenused for preparing carboanhydrase IX (Mn) antibodies in analogy for thepreparation of antibodies with different target molecule specificity.

Bacterial Expression

The person skilled in the art is aware of the way in which antibodies,antigen-binding fragments thereof or variants thereof can be producedwith the aid of bacterial expression.

Suitable expression vectors for bacterial expression of desired proteinsare constructed by insertion of a DNA sequence which encodes the desiredprotein within the functional reading frame together with suitabletranslation initiation and translation termination signals and with afunctional promoter. The vector comprises one or more phenotypicallyselectable markers and a replication origin in order to enable theretention of the vector and, if desired, the amplification thereofwithin the host. Suitable prokaryotic hosts for transformation includebut are not limited to E. coli, Bacillus subtilis, Salmonellatyphimurium and various species from the genus Pseudomonas,Streptomyces, and Staphylococcus. Bacterial vectors may be based, forexample, on bacteriophages, plasm ids, or phagemids. These vectors maycontain selectable markers and a bacterial replication origin, which arederived from commercially available plasmids. Many commerciallyavailable plasmids typically contain elements of the well-known cloningvector pBR322 (ATCC 37017). In bacterial systems, a number ofadvantageous expression vectors can be selected on the basis of theintended use of the protein to be expressed.

After transformation of a suitable host strain and growth of the hoststrain to an appropriate cell density, the selected promoter isde-reprimed/induced by suitable means (for example a change intemperature or chemical induction), and the cells are cultivated for anadditional period. The cells are typically harvested by centrifugationand if necessary digested in a physical manner or by chemical means, andthe resulting raw extract is retained for further purification.

Therefore, a further embodiment of the present invention is anexpression vector comprising a nucleic acid which encodes a novelantibody of the present invention.

Antibodies of the present invention or antigen-binding fragments thereofinclude naturally purified products, products which originate fromchemical syntheses, and products which are produced by recombinanttechnologies in prokaryotic hosts, for example E. coli, Bacillussubtilis, Salmonella typhimurium and various species from the genusPseudomonas, Streptomyces, and Staphylococcus, preferably E. coli.

Mammalian Cell Expression

The person skilled in the art is aware of the way in which antibodies,antigen-binding fragments thereof or variants thereof can be producedwith the aid of mammalian cell expression.

Preferred regulatory sequences for expression in mammalian cell hostsinclude viral elements which lead to high expression in mammalian cells,such as promoters and/or expression amplifiers derived fromcytomegalovirus (CMV) (such as the CMV promoter/enhancer), simian virus40 (SV40) (such as the SV40 promoter/enhancer), from adenovirus, (forexample the adenovirus major late promoter (AdMLP)) and from polyoma.

The expression of the antibodies may be constitutive or regulated (forexample induced by addition or removal of small molecule inductors suchas tetracycline in combination with the Tet system).

For further description of viral regulatory elements and sequencesthereof, reference is made, for example, to U.S. Pat. No. 5,168,062 byStinski, U.S. Pat. No. 4,510,245 by Bell et al. and U.S. Pat. No.4,968,615 by Schaffner et al. The recombinant expression vectors maylikewise include a replication origin and selectable markers (see, forexample, U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017). Suitableselectable markers include genes which impart resistance to substancessuch as G418, puromycin, hygromycin, blasticidin, zeocin/bleomycin, ormethotrexate, or selectable markers which lead to auxotrophy of a hostcell, such as glutamine synthetase (Bebbington et al., Biotechnology (NY). 1992 February; 10(2):169-75), when the vector has been introducedinto the cell.

For example, the dihydrofolate reductase (DHFR) gene imparts resistanceto methotrexate, the neo gene imparts resistance to G418, the bsd genefrom Aspergillus terreus imparts resistance to blasticidin, puromycinN-acetyltransferase imparts resistance to puromycin, the Sh ble geneproduct imparts resistance to zeocin, and resistance to hygromycin isimparted by the E. coli hygromycin resistance gene (hyg or hph).Selectable markers such as DHFR or glutamine synthetase are also helpfulfor amplification techniques in conjunction with MTX and MSX.

The transfection of an expression vector into a host cell can beexecuted with the aid of standard techniques, including byelectroporation, nucleofection, calcium phosphate precipitation,lipofection, polycation-based transfection such as polyethyleneimine(PEI)-based transfection and DEAE-dextran transfection.

Suitable mammalian host cells for the expression of antibodies,antigen-binding fragments thereof, or variants thereof include Chinesehamster ovary (CHO) cells such as CHO-K1, CHO-S, CHO-K1SV [includingDHFR-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad.Sci. USA 77:4216-4220 and Urlaub et al., Cell. 1983 June; 33(2):405-12,used with a DHFR-selectable marker, as described in R. J. Kaufman and P.A. Sharp (1982) Mol. Biol. 159:601-621, and other knockout cells, asdetailed in Fan et al., Biotechnol Bioeng. 2012 April; 109(4):1007-15),NSO myeloma cells, COS cells, HEK293 cells, HKB11 cells, BHK21 cells,CAP cells, EB66 cells, and SP2 cells.

The expression of antibodies, antigen-binding fragments thereof, orvariants thereof can also be effected in a transient or semi-stablemanner in expression systems such as HEK293, HEK293T, HEK293-EBNA,HEK293E, HEK293-6E, HEK293 Freestyle, HKB11, Expi293F, 293EBNALT75, CHOFreestyle, CHO-S, CHO-K1, CHO-K1SV, CHOEBNALT85, CHOS-XE, CHO-3E7 orCAP-T cells (for example like Durocher et al., Nucleic Acids Res. 2002Jan. 15; 30(2):E9)

In some embodiments, the expression vector is constructed in such a waythat the protein to be expressed is secreted into the cell culturemedium in which the host cells are growing. The antibodies, theantigen-binding fragments thereof, or the variants thereof can beobtained from the cell culture medium with the aid of proteinpurification methods known to those skilled in the art.

Purification

The antibodies, the antigen-binding fragments thereof, or the variantsthereof can be obtained and purified from recombinant cell cultures withthe aid of well-known methods, examples of which include ammoniumsulfate or ethanol precipitation, acid extraction, protein Achromatography, protein G chromatography, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography (HIC), affinity chromatography, hydroxyapatitechromatography and lectin chromatography. High-performance liquidchromatography (“HPLC”) can likewise be employed for purification. See,for example, Colligan, Current Protocols in Immunology, or CurrentProtocols in Protein Science, John Wiley & Sons, NY, N.Y., (1997-2001),e.g., Chapters 1, 4, 6, 8, 9, 10.

Antibodies of the present invention or antigen-binding fragmentsthereof, or variants thereof include naturally purified products,products from chemical synthesis methods and products which are producedwith the aid of recombinant techniques in prokaryotic or eukaryotic hostcells. Eukaryotic hosts include, for example, yeast cells, higher plantcells, insect cells and mammalian cells. Depending on the host cellchosen for the recombinant expression, the protein expressed may be inglycosylated or non-glycosylated form.

In a preferred embodiment, the antibody is purified (1) to an extent ofmore than 95% by weight, measured, for example, by the Lowry method, byUV-vis spectroscopy or by SDS capillary gel electrophoresis (for examplewith a Caliper LabChip GXII, GX 90 or Biorad Bioanalyzer instrument),and in more preferred embodiments more than 99% by weight, (2) to adegree suitable for determination of at least 15 residues of theN-terminal or internal amino acid sequence, or (3) to homogeneitydetermined by SDS-PAGE under reducing or non-reducing conditions withthe aid of Coomassie blue or preferably silver staining.

Usually, an isolated antibody is obtained with the aid of at least oneprotein purification step.

Anti-CD123 Antibodies

According to the invention, it is possible to use anti-CD123 antibodies.

The expression “anti-CD123 antibody” or “an antibody which bindsspecifically to CD123” relates to an antibody which binds the cancertarget molecule CD123 (IL3RA; NCBI-Gene ID: 3563; NCBI Referencesequence: NP_002174.1; Swiss-Prot: P26951; SEQ ID NO:111), preferablywith an affinity sufficient for a diagnostic and/or therapeuticapplication. In particular embodiments, the antibody binds CD123 with adissociation constant (K_(D)) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM,≤0.01 nM, or ≤0.001 nM.

Sun et al. (Sun et al., 1996, Blood 87(1)83-92) describe the generationand properties of the monoclonal antibody 7G3, which binds theN-terminal domain of IL-3Rα, CD123. U.S. Pat. No. 6,177,078 (Lopez)relates to the anti-CD123 antibody 7G3. A chimeric variant of thisantibody (CSL360) is described in WO 2009/070844, and a humanizedversion (CSL362) in WO 2012/021934. The sequence of the 7G3 antibody isdisclosed in EP2426148. This sequence constitutes the starting point forthe humanized antibodies obtained by CDR grafting.

An antibody which, after cell surface antigen binding, is internalizedparticularly well is the anti-CD123 antibody 12F1 disclosed by Kuo etal. (Kuo et al., 2009, Bioconjug Chem. 20(10):1975-82). The antibody12F1 binds with higher affinity to CD123 than the antibody 7G3 and,after cell surface antigen binding, is internalized markedly faster than7G3. Bispecific scFv immunofusion proteins based on 12F1 are disclosedin WO 2013/173820. Antibody TPP-6013 is a chimeric variant of 12F1.

The invention relates in particular to conjugates with antibodies orantigen-binding antibody fragments thereof or variants thereof derivedfrom the antibodies 7G3 (Sun et al., 1996, Blood 87(1):83-92) and 12F1(Kuo et al., 2009, Bioconjug Chem. 20(10):1975-82) originating from themouse, or to conjugates with antibodies or antigen-binding antibodyfragments thereof or variants thereof derived from the antibody 12F1(Kuo et al., 2009, Bioconjug Chem. 20(10):1975-82) originating from themouse.

Humanized variants of the murine 7G3 antibody and the murine 12F1antibody were generated by CDR grafting into a human framework andsubsequent optimization and are preferred examples in the context of thepresent invention.

Particular preference is given in the context of the present inventionto the anti-CD123 antibodies TPP-9476, TPP-8988, TPP-8987 and TPP-6013.

Anti-CXCR5 Antibodies

According to the invention, it is possible to use anti-CXCR5 antibodies.

The expression “anti-CXCR5 antibody” or “an antibody which bindsspecifically to CXCR5” relates to an antibody which binds the cancertarget molecule CXCR5 (NCBI Reference Sequence: NP_001707.1; SEQ ID NO:112), preferably with an affinity sufficient for a diagnostic and/ortherapeutic application. In particular embodiments, the antibody bindsCXCR5 with a dissociation constant (K_(D)) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM.

Examples of antibodies and antigen-binding fragments which bind to CXCR5are known to those skilled in the art and are described, for example, inEP2195023.

The hybridoma cells for the rat antibody RF8B2 (ACC2153) were purchasedfrom DSMZ and the sequence of the antibody was identified by standardmethods. This sequence constitutes the starting point for the humanizedantibodies obtained by CDR grafting.

Humanized variants of this antibody were generated by CDR grafting intogermline sequences.

These antibodies and antigen-binding fragments can be used in thecontext of this invention.

Particular preference is given in the context of the present inventionto the anti-CXCR5 antibodies TPP-9574 and TPP-9580.

Anti-B7H3 Antibodies

According to the invention, it is possible to use anti-B7H3 antibodies.

The expression “anti-B7H3 antibody” or “an antibody which bindsspecifically to B7H3” relates to an antibody which binds the cancertarget molecule B7H3 (NCBI Reference Sequence: NP_001019907.1 SEQ ID NO:113), preferably with an affinity sufficient for a diagnostic and/ortherapeutic application. In particular embodiments, the antibody bindsB7H3 with a dissociation constant (K_(D)) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM.

Examples of antibodies and antigen-binding fragments which bind to B7H3are known to those skilled in the art and are described, for example, inWO201109400, EP1773884 and WO2014061277. EP2121008 describes theanti-B7H3 antibody 8H9 and the CDR sequences thereof.

These antibodies and antigen-binding fragments can be used in thecontext of this invention.

A preferred embodiment of the anti-B7H3 antibodies was obtained byscreening an antibody phage display library for cells that expressrecombinant mouse B7H3 (mouse CD276; Gene ID: 102657) and human B7H3(human CD276; Gene ID: 80381). The antibodies obtained were transformedto the human IgG1 format. The anti-B7H3 antibody TPP-8382 is a preferredexample.

Particular preference is given in the context of the present inventionto the anti-B7H3 antibody TPP-8382.

Anti-TWEAKR Antibodies

According to the invention, it is possible to use anti-TWEAKRantibodies.

The expression “anti-TWEAKR antibody” or “an antibody which bindsspecifically to TWEAKR” relates to an antibody which binds the cancertarget molecule TWEAKR (NCBI Reference Sequence: NP_057723.1 SEQ ID NO:114), preferably with an affinity sufficient for a diagnostic and/ortherapeutic application. In particular embodiments, the antibody bindsTWEAKR with a dissociation constant (K_(D)) of ≤1 μM, ≤100 nM, ≤10 nM,≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM.

Examples of antibodies which bind to TWEAKR are disclosed, for example,in WO2009/020933(A2), WO2009/140177 (A2), WO 2014/1 98817 (A1) and WO2015/189143 (A1). These antibodies and antigen-binding fragments can beused in the context of this invention.

ITEM-4 is an anti-TWEAKR antibody which was described by Nakayama et al.(Nakayama, et al., 2003, Biochem Biophy Res Comm, 306:819-825).Humanized variants of this antibody based on CDR grafting are describedby Zhou et al. (Zhou et al., 2013, J Invest Dermatol. 133(4):1052-62)and in WO 2009/020933. These antibodies and antigen-binding fragmentscan be used in the context of this invention.

Particular preference is given in the context of the present inventionto the anti-TWEAKR antibodies TPP-7006 and TPP-7007. These are humanizedvariants of the antibody ITEM-4. These antibodies and antigen-bindingfragments can be used with preference in the context of this invention.

Anti-HER2 Antibodies:

According to the invention, it is possible to use anti-HER2 antibodies.

The expression “anti-HER2 antibody” or “an antibody which bindsspecifically to HER2” relates to an antibody which binds the cancertarget molecule HER2 (NCBI Reference Sequence: NP_004439.2 SEQ ID NO:115), preferably with an affinity sufficient for a diagnostic and/ortherapeutic application. In particular embodiments, the antibody bindsHER2 with a dissociation constant (K_(D)) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM.

An example of an antibody binding to the cancer target molecule Her2 istrastuzumab (Genentech). Trastuzumab is a humanized antibody used interalia for the treatment of breast cancer. In a particularly preferredembodiment, the anti-HER2 antibody is TPP-1015 (trastuzumab analogue).

Further examples of antibodies binding to HER2 are, in addition totrastuzumab (INN 7637, CAS No.: RN: 180288-69-1) and pertuzumab (CASNo.: 380610-27-5), the antibodies disclosed in WO 2009/123894-A2, WO200/8140603-A2 or in WO 2011/044368-A2. An example of an anti-HER2conjugate is trastuzumab-emtansine (INN-No. 9295). These antibodies andantigen-binding fragments can be used in the context of this invention.

Particular preference is given in the context of this invention to theanti-HER2 antibody TPP-1015 (analogous to trastuzumab).

Anti-EGFR Antibodies

According to the invention, it is possible to use anti-EGFR antibodies.

The expression “anti-EGFR antibody” or “an antibody which bindsspecifically to EGFR” relates to an antibody which binds the cancertarget molecule EGFR (NCBI Reference Sequence: NP_005219.2 SEQ ID NO:116), preferably with an affinity sufficient for a diagnostic and/ortherapeutic application. In particular embodiments, the antibody bindsEGFR with a dissociation constant (K_(D)) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM.

In a preferred embodiment, the anti-EGFR antibodies are selected fromthe group consisting of TPP-981, Cetuximab, panitumumab, nimotuzumab. Ina particularly preferred embodiment, the anti-EGFR antibody is TPP-981.

Further embodiments of EGFR antibodies are as follows:

-   -   zalutumumab/2F8/HuMax-EGFr, from Genmab A/S (WO 02/100348, WO        2004/056847, INN number 8605)    -   necitumumab/11F8, ImClone/IMC-11F8, from ImClone Systems Inc.        [Eli Lilly & Co] (WO 2005/090407 (EP 01735348-A1, US        2007/0264253-A1, U.S. Pat. No. 7,598,350, WO 2005/090407-A1),        INN number 9083)    -   matuzumab/anti-EGFR MAb, Merck KGaA/anti-EGFR MAb, Takeda/EMD        72000/EMD-6200/EMD-72000 and EMD-55900/MAb 425/monoclonal        antibody 425, from Merck KGaA/Takeda (WO 92/15683, INN number        8103 (matuzumab))    -   RG-7160/GA-201/GA201/R-7160/R7160/RG7160/RO-4858696/RO-5083945/R04858696/R05083945,        from Glycart Biotechnology AG (Roche Holding AG) (WO        2010/112413-A1, WO 2010/115554)    -   GT-MAB 5.2-GEX/CetuGEX, from Glycotope GmbH (WO 2008/028686-A2        (EP 01900750-A1, EP 01911766-A1, EP 02073842-A2, US        2010/0028947-A1)    -   ISU-101, from Isu Abxis Inc (ISU Chemical Co Ltd)/Scancell (WO        2008/004834-A1)    -   ABT-806/mAb-806/ch-806/anti-EGFR monoclonal antibody 806, from        Ludwig Institute for Cancer Research/Abbott/Life Science        Pharmaceuticals (WO 02/092771, WO 2005/081854 and WO        2009/023265)    -   SYM-004 (consists of two chimeric IgG1 antibodies (992 and        1024)), from Symphogen A/S (WO 2010/022736-A2)    -   MR1-1/MR1-1KDEL, from IVAX Corp (Teva Pharmaceutical Industries        Ltd) (Duke University), (patent: WO2001/062931-A2)    -   Antibody against the deletion mutant, EGFRvIII, from        Amgen/Abgenix (WO 2005/010151, U.S. Pat. No. 7,628,986)    -   SC-100, from Scancell Ltd (WO 01/088138-A1)    -   MDX-447/EMD 82633/BAB-447/H 447/MAb, EGFR, Medarex/Merck KgaA,        from Bristol-Myers Squibb (US)/Merck KGaA (DE)/Takeda (JP), (WO        91/05871, WO 92/15683)    -   anti-EGFR-Mab, from Xencor (WO 2005/056606)    -   DXL-1218/anti-EGFR monoclonal antibody (cancer), InNexus, from        InNexus Biotechnology Inc, Pharmaprojects PH048638

Anti-Carboanhydrase IX Antibodies

Examples of antibodies which bind the cancer target moleculecarbonanhydrase IX are described in WO 2007/070538-A2 (e.g. Claims1-16).

Anti-C4.4a Antibodies:

Examples of C4.4a antibodies and antigen-binding fragments are describedin WO 2012/143499 A2. The sequences of the antibodies are given in Table1 of WO 2012/143499 A2, where each row shows the respective CDR aminoacid sequences of the variable light chain or the variable heavy chainof the antibody listed in column 1.

Anti-CD20 Antibodies:

An example of an antibody binding to the cancer target molecule CD20 isrituximab (Genentech). Rituximab (CAS Number: 174722-31-7) is a chimericantibody used for the treatment of non-Hodgkin's lymphoma. Theseantibodies and antigen-binding fragments thereof can be used in thecontext of this invention.

Anti-CD52 Antibodies:

An example of an antibody binding to the cancer target molecule CD52 isalemtuzumab (Genzyme). Alemtuzumab (CAS Number: 216503-57-0) is ahumanized antibody used for the treatment of chronic lymphocyticleukaemia. These antibodies and antigen-binding fragments thereof can beused in the context of this invention.

Anti-Mesothelin Antibodies:

Examples of anti-mesothelin antibodies are described, for example, inWO2009/068204. All WO2009/068204 disclosed antibodies andantigen-binding fragments can be used in the context of the inventiondisclosed herein. More preferably, the antibody disclosed inWO2009/068204 is MF-T.

Anti-CD30 Antibodies

Examples of antibodies which bind the cancer target molecule CD30 andcan be used for the treatment of cancer, for example Hodgkin's lymphoma,are brentuximab, iratumumab and antibodies disclosed in WO 2008/092117,WO 2008/036688 or WO 2006/089232. An example of an anti-CD30 conjugateis brentuximab vedotin (INN No. 9144). These antibodies andantigen-binding fragments thereof can be used in the context of thisinvention.

Anti-CD22 Antibodies

Examples of antibodies which bind the cancer target molecule CD22 andcan be used for the treatment of cancer, for example lymphoma, areinotuzumab and epratuzumab. Examples of anti-CD22 conjugates areinotuzumab ozagamycin (INN No. 8574) or anti-CD22-MMAE andanti-CD22-MC-MMAE (CAS RN: 139504-50-0 and 474645-27-7, respectively).These antibodies and antigen-binding fragments thereof can be used inthe context of this invention.

Anti-CD33 Antibodies

Examples of antibodies which bind the cancer target molecule CD33 andcan be used for the treatment of cancer, for example leukaemia, aregemtuzumab and lintuzumab (INN 7580). An example of an anti-CD33conjugate is gemtuzumab-ozagamycin. These antibodies and antigen-bindingfragments can be used in the context of this invention.

Anti-NMB Antibodies

An example of an antibody which binds the cancer target molecule NMB andcan be used for the treatment of cancer, for example melanoma or breastcancer, is glembatumumab (INN 9199). An example of an anti-NMB conjugateis glembatumumab vedotin (CAS RN: 474645-27-7). These antibodies andantigen-binding fragments thereof can be used in the context of thisinvention.

Anti-CD56 Antibodies

An example of an antibody which binds the cancer target molecule CD56and can be used for the treatment of cancer, for example multiplemyeloma, small-cell lung carcinoma, MCC or ovarial carcinoma islorvotuzumab. An example of an anti-CD57 conjugate is lorvotuzumabmertansine (CAS RN: 139504-50-0). These antibodies and antigen-bindingfragments can be used in the context of this invention.

Anti-CD70 Antibodies

Examples of antibodies which bind the cancer target molecule CD70 andcan be used for the treatment of cancer, for example non-Hodgkin'slymphoma or renal cell cancer, are disclosed in WO 2007/038637-A2 and WO2008/070593-A2. An example of an anti-CD70 conjugate is SGN-75 (CD70MMAF). These antibodies and antigen-binding fragments can be used in thecontext of this invention.

Anti-CD74 Antibodies

An example of an antibody which binds the cancer target molecule CD74and can be used for treatment of cancer, for example multiple myeloma,is milatuzumab. An example of an anti-CD74 conjugate ismilatuzumab-doxorubicin (CAS RN: 23214-92-8). These antibodies andantigen-binding fragments can be used in the context of this invention.

Anti-CD19 Antibodies

An example of an antibody which binds the cancer target molecule CD19and can be used for the treatment of cancer, for example non-Hodgkin'slymphoma, is disclosed in WO 2008/031056-A2. Further antibodies andexamples of an anti-CD19 conjugate (SAR3419) are disclosed in WO2008/047242-A2. These antibodies and antigen-binding fragments thereofcan be used in the context of this invention.

Anti-Mucin Antibodies

Examples of antibodies which bind the cancer target molecule mucin-1 andcan be used for the treatment of cancer, for example non-Hodgkin'slymphoma, are clivatuzumab and the antibodies disclosed in WO2003/106495-A2, WO 2008/028686-A2. Examples of anti-mucin conjugates aredisclosed in WO 2005/009369-A2. These antibodies and antigen-bindingfragments thereof can be used in the context of this invention.

Anti-CD138 Antibodies

Examples of antibodies which bind the cancer target molecule CD138 andconjugates thereof, which can be used for the treatment of cancer, forexample multiple myeloma, are disclosed in WO 2009/080829-A1, WO2009/080830-A1. These antibodies and antigen-binding fragments thereofcan be used in the context of this invention.

Anti-Integrin-AlphaV Antibodies

Examples of antibodies which bind the cancer target molecule integrinalphaV and can be used for the treatment of cancer, for examplemelanoma, sarcoma or carcinoma, are intetumumab (CAS RN: 725735-28-4),abciximab (CAS RN: 143653-53-6), etaracizumab (CAS RN: 892553-42-3) andthe antibodies disclosed in U.S. Pat. No. 7,465,449, EP 719859-A1, WO2002/012501-A1 and WO2006/062779-A2. Examples of anti-integrin AlphaVconjugates are intetumumab-DM4 and other ADCs disclosed in WO2007/024536-A2. These antibodies and antigen-binding fragments thereofcan be used in the context of this invention.

Anti-TDGF1 Antibodies

Examples of antibodies which bind the cancer target molecule TDGF1 andcan be used for the treatment of cancer are the antibodies disclosed inWO 02/077033-A1, U.S. Pat. No. 7,318,924, WO 2003/083041-A2 and WO2002/088170-A2. Examples of anti-TDGF1 conjugates are disclosed in WO2002/088170-A2. These antibodies and antigen-binding fragments thereofcan be used in the context of this invention.

Anti-PSMA Antibodies

Examples of antibodies which bind the cancer target molecule PSMA andcan be used for the treatment of cancer, for example prostate carcinoma,are the antibodies disclosed in WO 97/35616-A1, WO 99/47554-A1, WO01/009192-A1 and WO2003/034903. Examples of anti-PSMA conjugates aredisclosed in WO 2009/026274-A1 and WO 2007/002222. These antibodies andantigen-binding fragments can be used in the context of this invention.

Anti-EPHA2 Antibodies

Examples of antibodies which bind the cancer target molecule EPHA2 andcan be used for preparing a conjugate and for the treatment of cancerare disclosed in WO 2004/091375-A2. These antibodies and antigen-bindingfragments can be used in the context of this invention.

Anti-SLC44A4 Antibodies

Examples of antibodies which bind the cancer target molecule SLC44A4 andcan be used for preparing a conjugate and for the treatment of cancer,for example pancreas or prostate carcinoma, are disclosed inWO2009/033094-A2 and US2009/0175796-A1. These antibodies andantigen-binding fragments thereof can be used in the context of thisinvention.

Anti-HLA-DOB Antibodies

An example of an antibody binding to the cancer target molecule HLA-DOBis the antibody Lym-1 (CAS RN: 301344-99-0) which can be used fortreatment of cancer, for example non-Hodgkin's lymphoma. Examples ofanti-HLA-DOB conjugates are disclosed, for example, in WO2005/081711-A2. These antibodies and antigen-binding fragments thereofcan be used in the context of this invention.

Anti-VTCN1 Antibodies

Examples of antibodies which bind the cancer target molecule VTCN1 andcan be used for preparing a conjugate and for the treatment of cancer,for example ovarial carcinoma, pancreas, lung or breast cancer, aredisclosed in WO 2006/074418-A2. These antibodies and antigen-bindingfragments thereof can be used in the context of this invention.

Anti-FGFR2 Antibodies

Examples of anti-FGFR2 antibodies and antigen-binding fragments aredescribed in WO2013076186. The sequences of the antibodies are shown inTable 9 and Table 10 of WO2013076186. Preference is given to antibodies,antigen-binding fragments and variants of the antibodies derived fromthe antibodies referred to as M048-D01 and M047-D08.

Preferred antibodies and antigen-binding antibody fragments forbinder-drug conjugates according to the invention

In this application, in the context of the binder-drug conjugates,reference is made to the following preferred antibodies as shown in thefollowing table: TPP-981, TPP-1015, TPP-6013, TPP-7006, TPP-7007,TPP-8382, TPP-8987, TPP-8988, TPP-9476, TPP-9574 and TPP-9580.

TABLE Protein sequences of the antibodies: SEQ ID SEQ ID SEQ ID SEQ IDSEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: SEQ ID NO: Antibody NO: NO: NO:NO: NO: NO: NO: NO: IgG heavy IgG light TPP-XXX Antigen VH H-CDR1 H-CDR2H-CDR3 VI L-CDR1 L-CDR2 L-CDR3 chain chain TPP-981 EGFR  1  2  3  4  5 6  7  8  9  10 TPP-1015 HER2 11 12 13 14 15 16 17 18 19  20 TPP-6013CD123 21 22 23 24 25 26 27 28 29  30 TPP-7006 TWEAKR 31 32 33 34 35 3637 38 39  40 TPP-7007 TWEAKR 41 42 43 44 45 46 47 48 49  50 TPP-8382B7H3 51 52 53 54 55 56 57 58 59  60 TPP-8987 CD123 61 62 63 64 65 66 6768 69  70 TPP-8988 CD123 71 72 73 74 75 76 77 78 79  80 TPP-9476 CD12381 82 83 84 85 86 87 88 89  90 TPP-9574 CXCR5 91 92 93 94 95 96 97 98 99100 TPP-9580 CXCR5 101  102  103  104  105  106  107  108  109  110

TPP-981, TPP-1015, TPP-6013, TPP-7006, TPP-7007, TPP-8382, TPP-8987,TPP-8988, TPP-9476, TPP-9574 and TPP-9580 are antibodies comprising oneor more of the CDR sequences specified in the above table (H-CDR1,H-CDR2, H-CDR3, L-CDR1, L-CDR2, L-CDR3) in the variable region of theheavy chain (VH) or the variable region of the light chain (VL).Preferably, the antibodies comprise the specified variable region of theheavy chain (VH) and/or the variable region of the light chain (VL).Preferably, the antibodies comprise the specified region of the heavychain (IgG heavy chain) and/or the specified region of the light chain(IgG light chain).

TPP-981 is an anti-EGFR antibody comprising a variable region of theheavy chain (VH) comprising the variable CDR1 sequence of the heavychain (H-CDR1), as shown by SEQ ID NO: 2, the variable CDR2 sequence ofthe heavy chain (H-CDR2), as shown by SEQ ID NO: 3 and the variable CDR3sequence of the heavy chain (H-CDR3), as shown by SEQ ID NO: 4, and avariable region of the light chain (VL) comprising the variable CDR1sequence of the light chain (L-CDR1), as shown by SEQ ID NO: 6, thevariable CDR2 sequence of the light chain (L-CDR2), as shown by SEQ IDNO: 7 and the variable CDR3 sequence of the light chain (L-CDR3), asshown by SEQ ID NO: 8.

TPP-1015 is an anti-HER2 antibody comprising a variable region of theheavy chain (VH) comprising the variable CDR1 sequence of the heavychain (H-CDR1), as shown by SEQ ID NO: 12, the variable CDR2 sequence ofthe heavy chain (H-CDR2), as shown by SEQ ID NO: 13 and the variableCDR3 sequence of the heavy chain (H-CDR3), as shown by SEQ ID NO: 14,and a variable region of the light chain (VL) comprising the variableCDR1 sequence of the light chain (L-CDR1), as shown by SEQ ID NO: 16,the variable CDR2 sequence of the light chain (L-CDR2), as shown by SEQID NO: 17 and the variable CDR3 sequence of the light chain (L-CDR3), asshown by SEQ ID NO: 18.

TPP-6013 is an anti-CD123 antibody comprising a variable region of theheavy chain (VH) comprising the variable CDR1 sequence of the heavychain (H-CDR1), as shown by SEQ ID NO: 22, the variable CDR2 sequence ofthe heavy chain (H-CDR2), as shown by SEQ ID NO: 23 and the variableCDR3 sequence of the heavy chain (H-CDR3), as shown by SEQ ID NO: 24,and a variable region of the light chain (VL) comprising the variableCDR1 sequence of the light chain (L-CDR1), as shown by SEQ ID NO: 26,the variable CDR2 sequence of the light chain (L-CDR2), as shown by SEQID NO: 27 and the variable CDR3 sequence of the light chain (L-CDR3), asshown by SEQ ID NO: 28.

TPP-7006 is an anti-TWEAKR antibody comprising a variable region of theheavy chain (VH) comprising the variable CDR1 sequence of the heavychain (H-CDR1), as shown by SEQ ID NO: 32, the variable CDR2 sequence ofthe heavy chain (H-CDR2), as shown by SEQ ID NO: 33 and the variableCDR3 sequence of the heavy chain (H-CDR3), as shown by SEQ ID NO: 34,and a variable region of the light chain (VL) comprising the variableCDR1 sequence of the light chain (L-CDR1), as shown by SEQ ID NO: 36,the variable CDR2 sequence of the light chain (L-CDR2), as shown by SEQID NO: 37 and the variable CDR3 sequence of the light chain (L-CDR3), asshown by SEQ ID NO: 38.

TPP-7007 is an anti-TWEAKR antibody comprising a variable region of theheavy chain (VH) comprising the variable CDR1 sequence of the heavychain (H-CDR1), as shown by SEQ ID NO: 42, the variable CDR2 sequence ofthe heavy chain (H-CDR2), as shown by SEQ ID NO: 43 and the variableCDR3 sequence of the heavy chain (H-CDR3), as shown by SEQ ID NO: 44,and a variable region of the light chain (VL) comprising the variableCDR1 sequence of the light chain (L-CDR1), as shown by SEQ ID NO: 46,the variable CDR2 sequence of the light chain (L-CDR2), as shown by SEQID NO: 47 and the variable CDR3 sequence of the light chain (L-CDR3), asshown by SEQ ID NO: 48.

TPP-8382 is an anti-B7H3 antibody comprising a variable region of theheavy chain (VH) comprising the variable CDR1 sequence of the heavychain (H-CDR1), as shown by SEQ ID NO: 52, the variable CDR2 sequence ofthe heavy chain (H-CDR2), as shown by SEQ ID NO: 53 and the variableCDR3 sequence of the heavy chain (H-CDR3), as shown by SEQ ID NO: 54,and a variable region of the light chain (VL) comprising the variableCDR1 sequence of the light chain (L-CDR1), as shown by SEQ ID NO: 56,the variable CDR2 sequence of the light chain (L-CDR2), as shown by SEQID NO: 57 and the variable CDR3 sequence of the light chain (L-CDR3), asshown by SEQ ID NO: 58.

TPP-8987 is an anti-CD123 antibody comprising a variable region of theheavy chain (VH) comprising the variable CDR1 sequence of the heavychain (H-CDR1), as shown by SEQ ID NO: 62, the variable CDR2 sequence ofthe heavy chain (H-CDR2), as shown by SEQ ID NO: 63 and the variableCDR3 sequence of the heavy chain (H-CDR3), as shown by SEQ ID NO: 64,and a variable region of the light chain (VL) comprising the variableCDR1 sequence of the light chain (L-CDR1), as shown by SEQ ID NO: 66,the variable CDR2 sequence of the light chain (L-CDR2), as shown by SEQID NO: 67 and the variable CDR3 sequence of the light chain (L-CDR3), asshown by SEQ ID NO: 68.

TPP-8988 is an anti-CD123 antibody comprising a variable region of theheavy chain (VH) comprising the variable CDR1 sequence of the heavychain (H-CDR1), as shown by SEQ ID NO: 72, the variable CDR2 sequence ofthe heavy chain (H-CDR2), as shown by SEQ ID NO: 73 and the variableCDR3 sequence of the heavy chain (H-CDR3), as shown by SEQ ID NO: 74,and a variable region of the light chain (VL) comprising the variableCDR1 sequence of the light chain (L-CDR1), as shown by SEQ ID NO: 76,the variable CDR2 sequence of the light chain (L-CDR2), as shown by SEQID NO: 77 and the variable CDR3 sequence of the light chain (L-CDR3), asshown by SEQ ID NO: 78.

TPP-9476 is an anti-CD123 antibody comprising a variable region of theheavy chain (VH) comprising the variable CDR1 sequence of the heavychain (H-CDR1), as shown by SEQ ID NO: 82, the variable CDR2 sequence ofthe heavy chain (H-CDR2), as shown by SEQ ID NO: 83 and the variableCDR3 sequence of the heavy chain (H-CDR3), as shown by SEQ ID NO: 84,and a variable region of the light chain (VL) comprising the variableCDR1 sequence of the light chain (L-CDR1), as shown by SEQ ID NO: 86,the variable CDR2 sequence of the light chain (L-CDR2), as shown by SEQID NO: 87 and the variable CDR3 sequence of the light chain (L-CDR3), asshown by SEQ ID NO: 88.

TPP-9574 is an anti-CXCR5 antibody comprising a variable region of theheavy chain (VH) comprising the variable CDR1 sequence of the heavychain (H-CDR1), as shown by SEQ ID NO: 92, the variable CDR2 sequence ofthe heavy chain (H-CDR2), as shown by SEQ ID NO: 93 and the variableCDR3 sequence of the heavy chain (H-CDR3), as shown by SEQ ID NO: 94,and a variable region of the light chain (VL) comprising the variableCDR1 sequence of the light chain (L-CDR1), as shown by SEQ ID NO: 96,the variable CDR2 sequence of the light chain (L-CDR2), as shown by SEQID NO: 97 and the variable CDR3 sequence of the light chain (L-CDR3), asshown by SEQ ID NO: 98.

TPP-9580 is an anti-CXCR5 antibody comprising a variable region of theheavy chain (VH) comprising the variable CDR1 sequence of the heavychain (H-CDR1), as shown by SEQ ID NO: 102, the variable CDR2 sequenceof the heavy chain (H-CDR2), as shown by SEQ ID NO: 103 and the variableCDR3 sequence of the heavy chain (H-CDR3), as shown by SEQ ID NO: 104,and a variable region of the light chain (VL) comprising the variableCDR1 sequence of the light chain (L-CDR1), as shown by SEQ ID NO: 106,the variable CDR2 sequence of the light chain (L-CDR2), as shown by SEQID NO: 107 and the variable CDR3 sequence of the light chain (L-CDR3),as shown by SEQ ID NO: 108.

TPP-981 is an anti-EGFR antibody comprising preferably a variable regionof the heavy chain (VH) as shown in SEQ ID NO: 1 and a variable regionof the light chain (VL) as shown in SEQ ID NO: 5.

TPP-1015 is an anti-HER2 antibody comprising preferably a variableregion of the heavy chain (VH) as shown in SEQ ID NO: 11 and a variableregion of the light chain (VL) as shown in SEQ ID NO: 15.

TPP-6013 is an anti-CD123 antibody comprising preferably a variableregion of the heavy chain (VH) as shown in SEQ ID NO: 21 and a variableregion of the light chain (VL) as shown in SEQ ID NO: 25.

TPP-7006 is an anti-TWEAKR antibody comprising preferably a variableregion of the heavy chain (VH) as shown in SEQ ID NO: 31 and a variableregion of the light chain (VL) as shown in SEQ ID NO: 35.

TPP-7007 is an anti-TWEAKR antibody comprising preferably a variableregion of the heavy chain (VH) as shown in SEQ ID NO: 41 and a variableregion of the light chain (VL) as shown in SEQ ID NO: 45.

TPP-8382 is an anti-B7H3 antibody comprising preferably a variableregion of the heavy chain (VH) as shown in SEQ ID NO: 51 and a variableregion of the light chain (VL) as shown in SEQ ID NO: 55.

TPP-8987 is an anti-CD123 antibody comprising preferably a variableregion of the heavy chain (VH) as shown in SEQ ID NO: 61 and a variableregion of the light chain (VL) as shown in SEQ ID NO: 65.

TPP-8988 is an anti-CD123 antibody comprising preferably a variableregion of the heavy chain (VH) as shown in SEQ ID NO: 71 and a variableregion of the light chain (VL) as shown in SEQ ID NO: 75.

TPP-9476 is an anti-CD123 antibody comprising preferably a variableregion of the heavy chain (VH) as shown in SEQ ID NO: 81 and a variableregion of the light chain (VL) as shown in SEQ ID NO: 85.

TPP-9574 is an anti-CXCR5 antibody comprising preferably a variableregion of the heavy chain (VH) as shown in SEQ ID NO: 91 and a variableregion of the light chain (VL) as shown in SEQ ID NO: 95.

TPP-9580 is an anti-CXCR5 antibody comprising preferably a variableregion of the heavy chain (VH) as shown in SEQ ID NO: 101 and a variableregion of the light chain (VL) as shown in SEQ ID NO: 105.

TPP-981 is an anti-EGFR antibody comprising preferably a region of theheavy chain as shown in SEQ ID NO: 9 and a region of the light chain asshown in SEQ ID NO: 10.

TPP-1015 is an anti-HER2 antibody comprising preferably a region of theheavy chain as shown in SEQ ID NO: 19 and a region of the light chain asshown in SEQ ID NO: 20.

TPP-6013 is an anti-CD123 antibody comprising preferably a region of theheavy chain as shown in SEQ ID NO: 29 and a region of the light chain asshown in SEQ ID NO: 30.

TPP-7006 is an anti-TWEAKR antibody comprising preferably a region ofthe heavy chain as shown in SEQ ID NO: 39 and a region of the lightchain as shown in SEQ ID NO: 40.

TPP-7007 is an anti-TWEAKR antibody comprising preferably a region ofthe heavy chain as shown in SEQ ID NO: 49 and a region of the lightchain as shown in SEQ ID NO: 50.

TPP-8382 is an anti-B7H3 antibody comprising preferably a region of theheavy chain as shown in SEQ ID NO: 59 and a region of the light chain asshown in SEQ ID NO: 60.

TPP-8987 is an anti-CD123 antibody comprising preferably a region of theheavy chain as shown in SEQ ID NO: 69 and a region of the light chain asshown in SEQ ID NO: 70.

TPP-8988 is an anti-CD123 antibody comprising preferably a region of theheavy chain as shown in SEQ ID NO: 79 and a region of the light chain asshown in SEQ ID NO: 80.

TPP-9476 is an anti-CD123 antibody comprising preferably a region of theheavy chain as shown in SEQ ID NO: 89 and a region of the light chain asshown in SEQ ID NO: 90.

TPP-9574 is an anti-CXCR5 antibody comprising preferably a region of theheavy chain as shown in SEQ ID NO: 99 and a region of the light chain asshown in SEQ ID NO: 100.

TPP-9580 is an anti-CXCR5 antibody comprising preferably a region of theheavy chain as shown in SEQ ID NO: 109 and a region of the light chainas shown in SEQ ID NO: 110.

Isotopes, Salts, Solvates, Isotopic Variants

The present invention also encompasses all suitable isotopic variants ofthe compounds of the invention. An isotopic variant of a compound of theinvention is understood here to mean a compound in which at least oneatom within the compound of the invention has been exchanged for anotheratom of the same atomic number, but with a different atomic mass fromthe atomic mass which usually or predominantly occurs in nature.Examples of isotopes which can be incorporated into a compound of theinvention are those of hydrogen, carbon, nitrogen, oxygen, phosphorus,sulfur, fluorine, chlorine, bromine and iodine, such as ²H (deuterium),³H (tritium), ¹³C, ¹⁴C, ¹⁵N, ¹⁷O, ¹⁸O, ³²P, ³³P, ³³S, ³⁴S, ³⁵S, ³⁶S,¹⁸F, ³⁶Cl, ⁸²Br, ¹²³I, ¹²⁴I, ¹²⁹I and ¹³¹I. Particular isotopic variantsof a compound according to the invention, especially those in which oneor more radioactive isotopes have been incorporated, may be beneficial,for example, for the examination of the mechanism of action or of theactive ingredient distribution in the body; due to the comparativelyeasy preparability and detectability, especially compounds labelled with³H or ¹⁴C isotopes are suitable for this purpose. In addition, theincorporation of isotopes, for example of deuterium, may lead toparticular therapeutic benefits as a consequence of greater metabolicstability of the compound, for example an extension of the half-life inthe body or a reduction in the active dose required; such modificationsof the compounds according to the invention may therefore in some casesalso constitute a preferred embodiment of the present invention.Isotopic variants of the compounds according to the invention can beprepared by the processes known to those skilled in the art, for exampleby the methods described further down and the procedures described inthe working examples, by using corresponding isotopic modifications ofthe respective reagents and/or starting compounds.

Preferred salts in the context of the present invention arephysiologically acceptable salts of the compounds of the invention. Alsoencompassed are salts which are not themselves suitable forpharmaceutical applications but can be used, for example, for isolationor purification of the compounds of the invention.

Physiologically acceptable salts of the compounds according to theinvention include acid addition salts of mineral acids, carboxylic acidsand sulfonic acids, for example salts of hydrochloric acid, hydrobromicacid, sulfuric acid, phosphoric acid, methanesulfonic acid,ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid,naphthalenedisulfonic acid, acetic acid, trifluoroacetic acid, propionicacid, lactic acid, tartaric acid, malic acid, citric acid, fumaric acid,maleic acid and benzoic acid.

Physiologically acceptable salts of the compounds according to theinvention also include salts of conventional bases, by way of exampleand with preference alkali metal salts (e.g. sodium and potassiumsalts), alkaline earth metal salts (e.g. calcium and magnesium salts)and ammonium salts derived from ammonia or organic amines having 1 to 16carbon atoms, by way of example and with preference ethylamine,diethylamine, triethylamine, ethyldiisopropylamine, monoethanolamine,diethanolamine, triethanolamine, dicyclohexylamine,dimethylaminoethanol, procaine, dibenzylamine, N-methylpiperidine,N-methylmorpholine, arginine, lysine and 1,2-ethylenediamine.

Solvates in the context of the invention are described as those forms ofthe compounds of the invention which form a complex in the solid orliquid state by coordination with solvent molecules. Hydrates are aspecific form of the solvates in which the coordination is with water.Solvates preferred in the context of the present invention are hydrates.

Therapeutic Use

The hyper-proliferative diseases, for the treatment of which thecompounds according to the invention may be employed, include inparticular the group of cancer and tumour diseases. In the context ofthe present invention, these are understood to mean especially thefollowing diseases, but without any limitation thereto: mammarycarcinomas and mammary tumours (mammary carcinomas including ductal andlobular forms, also in situ), tumours of the respiratory tract(small-cell and non-small cell carcinoma, bronchial carcinoma), cerebraltumours (e.g. of the brain stem and of the hypothalamus, astrocytoma,ependymoma, glioblastoma, glioma, medulloblastoma, meningioma andneuro-ectodermal and pineal tumours), tumours of the digestive organs(carcinomas of the oesophagus, stomach, gall bladder, small intestine,large intestine, rectum and anal carcinomas), liver tumours (inter aliahepatocellular carcinoma, cholangiocarcinoma and mixed hepatocellularcholangiocarcinoma), tumours of the head and neck region (larynx,hypopharynx, nasopharynx, oropharynx, lips and oral cavity carcinomas,oral melanomas), skin tumours (basaliomas, spinaliomas, squamous cellcarcinomas, Kaposi's sarcoma, malignant melanoma, non-melanomatous skincancer, Merkel cell skin cancer, mast cell tumours), tumours ofconnective tissue (inter alia soft tissue sarcomas, osteosarcomas,malignant fibrous histiocytomas, chondrosarcomas, fibrosarcomas,haemangiosarcomas, leiomyosarcomas, liposarcomas, lymphosarcomas andrhabdomyosarcomas), tumours of the eyes (inter alia intraocular melanomaand retinoblastoma), tumours of the endocrine and exocrine glands (e.g.of the thyroid and parathyroid glands, pancreas and salivary glandcarcinomas, adenocarcinomas), tumours of the urinary tract (tumours ofthe bladder, penis, kidney, renal pelvis and ureter) and tumours of thereproductive organs (carcinomas of the endometrium, cervix, ovary,vagina, vulva and uterus in women and carcinomas of the prostate andtestes in men). These also include proliferative diseases of the blood,the lymph system and the spinal cord, in solid form and as circulatingcells, such as leukaemias, lymphomas and myeloproliferative diseases,for example acute myeloid, acute lymphoblastic, chronic lymphocytic,chronic myelogenous and hairy cell leukaemia, and AIDS-correlatedlymphomas, Hodgkin's lymphomas, non-Hodgkin's lymphomas, cutaneous Tcell lymphomas, Burkitt's lymphomas and lymphomas in the central nervoussystem.

These well-characterized diseases in humans can also occur with acomparable aetiology in other mammals and can likewise be treated therewith the compounds of the present invention.

The binder- or antibody-drug conjugates (ADCs) described herein anddirected against CD123 can preferably be used for the treatment ofCD123-expressing disorders, such as CD123-expressing cancer diseases.Typically, such cancer cells exhibit measurable amounts of CD123measured at the protein (e.g. using an immunoassay) or RNA level. Someof these cancer tissues show an elevated level of CD123 compared tonon-cancerogenous tissue of the same type, preferably measured in thesame patient. Optionally, the CD123 content is measured prior to thestart of the cancer treatment with an antibody-drug conjugate (ADC)according to the invention (patient stratification). The binder-drugconjugates (ADCs) directed against CD123 can preferably be used for thetreatment of CD123-expressing disorders, such as CD123-expressing cancerdiseases, such as tumours of the haematopoietic and lymphatic tissue orhaematopoietic and lymphatic malignant tumours. Examples of cancerdiseases associated with CD123 expression include myeloid diseases suchas acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS).Other types of cancer include B-cell acute lymphoblastic leukaemia(B-ALL), hairy cell leukaemia, blastic plasmacytoid dendritic cellneoplasm (BPDCN), Hodgkin's lymphoma, immature T-cell acutelymphoblastic leukaemia (immature T-ALL), Burkitt's lymphoma, follicularlymphoma, chronic lymphocytic leukaemia (CLL), mantle cell lymphoma(MCL). Methods of the described invention comprise the treatment ofpatients suffering from CD123-expressing cancer, the method comprisingthe administration of an antibody-drug conjugate (ADC) according to theinvention.

The treatment of the cancer diseases mentioned above with the compoundsaccording to the invention comprises both a treatment of the solidtumours and a treatment of metastasizing or circulating forms thereof.

In the context of this invention, the term “treatment” or “treat” isused in the conventional sense and means attending to, caring for andnursing a patient with the aim of combating, reducing, attenuating oralleviating a disease or health abnormality, and improving the livingconditions impaired by this disease, as, for example, in the event of acancer.

The present invention thus further provides for the use of the compoundsof the invention for treatment and/or prevention of disorders,especially of the aforementioned disorders.

The present invention further provides for the use of the compounds ofthe invention for production of a medicament for treatment and/orprevention of disorders, especially of the aforementioned disorders.

The present invention further provides for the use of the compounds ofthe invention in a method for treatment and/or prevention of disorders,especially of the aforementioned disorders.

The present invention further provides a method of treatment and/orprevention of disorders, especially of the aforementioned disorders,using an effective amount of at least one of the compounds of theinvention.

The compounds of the invention can be used alone or, if required, incombination with one or more other pharmacologically active substances,provided that this combination does not lead to undesirable andunacceptable side effects. The present invention therefore furtherprovides medicaments comprising at least one of the compounds of theinvention and one or more further drugs, especially for treatment and/orprevention of the aforementioned disorders.

For example, the compounds of the present invention can be combined withknown anti-hyper-proliferative, cytostatic, cytotoxic orimmunotherapeutic substances for the treatment of cancer diseases.Examples of suitable combination drugs include:

131 I-chTNT, abarelix, abiraterone, aclarubicin, adalimumab,ado-trastuzumab emtansine, afatinib, aflibercept, aldesleukin,alemtuzumab, alendronic acid, alitretinoin, altretamine, amifostine,aminoglutethimide, hexyl-5-aminolevulinate, amrubicin, amsacrine,anastrozole, ancestim, anethole dithiolethione, anetumab ravtansine,angiotensin II, antithrombin III, aprepitant, arcitumomab, arglabin,arsenic trioxide, asparaginase, atezolizumab, avelumab, axitinib,azacitidine, belotecan, bendamustine, besilesomab, belinostat,bevacizumab, bexarotene, bicalutamide, bisantrene, bleomycin,blinatumomab, bortezomib, buserelin, bosutinib, brentuximab vedotin,busulfan, cabazitaxel, cabozantinib, calcitonin, calcium folinate,calcium levofolinate, capecitabine, capromab, carbomazepine,carboplatin, carboquon, carfilzomib, carmofur, carmustine, catumaxomab,celecoxib, celmoleukin, ceritinib, cetuximab, chlorambucil,chlormadinone, chlormethine, cidofovir, cinacalcet, cisplatin,cladribine, clodronic acid, clofarabine, cobimetinib, copanlisib,crisantaspase, crizotinib, cyclophosphamide, cyproterone, cytarabine,dacarbazine, dactinomycin, daratumumab, dabrafenib, darolutamide,dasatinib, daunorubicin, decitabine, degarelix, denileukin-diftitox,denosumab, depreotide, deslorelin, dexrazoxane, dibrospidium chloride,dianhydrogalactitol, diclofenac, docetaxel, dolasetron, doxifluridine,doxorubicin, doxorubicin+estrone, dronabinol, durvalumab, edrecolomab,elliptinium acetate, endostatin, enocitabine, enzalutamide, epacadostat,epirubicin, epitiostanol, epoetin-alfa, epoetin-beta, epoetin-zeta,eptaplatin, eribulin, erlotinib, esomeprazole, estramustine, etoposide,ethylnyl oestradiol, everolimus, exemestane, fadrozole, fentanyl,fluoxymesterone, floxuridine, fludarabine, fluorouracil, flutamide,folic acid, formestane, fosaprepitant, fotemustine, fulvestrant,gadobutrol, gadoteridol, gadoteric acid meglumine salt, gadoversetamide,gadoxetic acid disodium salt (gd-EOB-DTPA disodium salt), galliumnitrate, ganirelix, gefitinib, gemcitabine, gemtuzumab, glucarpidase,glutoxim, goserelin, granisetron, granulocyte colony stimulating factor(G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF),histamine dihydrochloride, histrelin, hydroxycarbamide, I-125 seeds,ibandronic acid, ibritumomab-tiuxetan, ibrutinib, idarubicin,ifosfamide, imatinib, imiquimod, improsulfan, indisetron, incadronicacid, ingenol mebutate, interferon-alfa, interferon-beta,interferon-gamma, iobitridol, iobenguane (123I), iomeprol, ipilimumab,irinotecan, itraconazole, ixabepilone, ixazomib, lanreotide,lansoprazole, lapatinib, lasocholine, lenalidomide, lenvatinib,lenograstim, lentinan, letrozole, leuprorelin, levamisole,levonorgestrel, levothyroxin-sodium, lipegfilgrastim, lisuride,lobaplatin, lomustine, lonidamine, masoprocol, medroxyprogesteron,megestrol, melarsoprol, melphalan, mepitiostan, mercaptopurine, mesna,methadone, methotrexate, methoxsalen, methylaminolevulinate,methylprednisolone, methyltestosterone, metirosine, mifamurtide,miltefosine, miriplatin, mitobronitol, mitoguazone, mitolactol,mitomycin, mitotan, mitoxantrone, mogamulizumab, molgramostim,mopidamol, morphine hydrochloride, morphine sulfate, nabilone,nabiximols, nafarelin, naloxone+pentazocine, naltrexone, nartograstim,necitumumab, nedaplatin, nelarabine, neridronic acid,netupitant/palonosetrone, nivolumab, nivolumab, pentetreotide,nilotinib, nilutamide, nimorazole, nimotuzumab, nimustine, nintedanib,nitracrine, nivolumab, obinutuzumab, octreotide, ofatumumab, olaparib,olaratumab, omacetaxin mepesuccinate, omeprazole, ondansetron, orgotein,orilotimod, osimertinib, oxaliplatin, oxycodone, oxymetholone,ozogamicin, p53 gene therapy, paclitaxel, palbociclib, palifermine,palladium-103 seed, palonosetron, pamidronic acid, panitumumab,panobinostat, pantoprazole, pazopanib, pegaspargase, pembrolizumab,Peg-interferon alfa-2b, pembrolizumab, pemetrexed, pentostatin,peplomycin, perflubutane, perfosfamide, pertuzumab, picibanil,pilocarpine, pirarubicin, pixantron, plerixafor, plicamycin, poliglusam,polyoestradiol phosphate, polyvinylpyrrolidone+sodium hyaluronate,polysaccharide-K, pomalidomide, ponatinib, porfimer-sodium,pralatrexate, prednimustine, prednisone, procarbazine, procodazole,propranolol, quinagolide, rabeprazole, racotumomab, radium-223 chloride,radotinib, raloxifene, raltitrexed, ramosetron, ramucirumab,ranimustine, rasburicase, razoxan, refametinib, regorafenib, risedronicacid, rhenium-186 etidronate, rituximab, rogaratinib, rolapitant,romidepsin, romurtid, roniciclib, samarium (153Sm) lexidronam,satumomab, secretin, siltuximab, sipuleucel-T, sizofiran, sobuzoxane,sodium glycididazole, sonidegib, sorafenib, stanozolol, streptozocin,sunitinib, talaporf in, talimogen laherparepvec, tamibarotene,tamoxifen, tapentadol, tasonermin, teceleukin, technetium (99mTc)nofetumomab merpentan, 99mTc-HYNIC-[Tyr3]-octreotide, tegafur,tegafur+gimeracil+oteracil, temoporfin, temozolomide, temsirolimus,teniposide, testosterone, tetrofosmin, thalidomide, thiotepa,thymalfasin, thyrotropin alfa, tioguanine, tocilizumab, topotecan,toremifene, tositumomab, trabectedin, trametinib, tramadol, trastuzumab,treosulfan, tretinoin, trifluridine+tipiracil, trametinib, trilostane,triptorelin, trofosfamide, thrombopoietin, ubenimex, valrubicin,vandetanib, vapreotide, vatalanib, vemurafenib, vinblastine,vincristine, vindesine, vinflunine, vinorelbine, vismodegib, vorinostat,yttrium-90 glass microbeads, zinostatin, zinostatin stimalamer,zoledronic acid, zorubicin

In addition, the compounds of the present invention can be combined, forexample, with binders (e.g. antibodies) which, by way of example, canbind to the following targets: OX-40, CD137/4-1BB, DR3, IDO1/1DO2,LAG-3, CD40.

Since a non-cell-permeable toxophore metabolite of a binder-drugconjugate (ADC) should have no damaging effect on the cells of theadaptive immune system, the invention furthermore provides thecombination of a binder-drug conjugate (ADC) according to the inventionwith a cancer immunotherapy for use in the treatment of cancer ortumours. The intrinsic mechanism of action of cytotoxic binder-drugconjugates comprises the direct triggering of cell death of the tumourcells and thus the release of tumour antigens which may stimulate animmune response. Furthermore, there are indications that the KSPinhibitor toxophore class induces markers of immunogenic cell death(ICD) in vitro. Thus, the combination of the binder-drug conjugates(ADCs) of the present invention with one or more therapeutic approachesof cancer immunotherapy or with one or more active compounds, preferablyantibodies, directed against a molecular target of cancer immunotherapyrepresents a preferred method for treating cancer or tumours. i)Examples of therapeutic approaches of cancer immunotherapy compriseimmunomodulatory monoclonal antibodies and low-molecular weightsubstances directed against targets of cancer immunotherapy, vaccines,CAR T cells, bispecific T-cell-recruiting antibodies, oncolyticalviruses, cell-based vaccination approaches. ii) Examples of selectedtargets of cancer immunotherapy suitable for immunemodulatory monoclonalantibodies comprise CTLA-4, PD-1/PDL-1, OX-40, CD137, DR3, IDO1,IDO2,TDO2, LAG-3, TIM-3 CD40, ICOS/ICOSL, TIGIT; GITR/GITRL, VISTA,CD70, CD27, HVEM/BTLA, CEACAM1, CEACAM6, ILDR2, CD73, CD47, B7H3, TLRs.Accordingly, combination of a binder-drug conjugate (ADC) according tothe invention with cancer immunotherapy could, on the one hand, rendertumours with weak immunogenic properties more immunogenic and enhancethe effectiveness of cancer immunotherapy, and furthermore unfoldlong-lasting therapeutic action.

In addition, the compounds according to the invention can also be usedin combination with radiotherapy and/or surgical intervention.

Generally, the following aims can be pursued with the combination ofcompounds of the present invention with other cytostatically,cytotoxically or immunotherapeutically active agents:

-   -   improved efficacy in slowing the growth of a tumour, in reducing        its size or even in completely eliminating it, compared with        treatment with an individual active ingredient;    -   the possibility of using the chemotherapeutics used in a lower        dosage than in the case of monotherapy;    -   the possibility of a more tolerable therapy with fewer side        effects compared with individual administration;    -   the possibility of treatment of a broader spectrum of neoplastic        disorders;    -   the achievement of a higher rate of response to the therapy;    -   a longer survival time of the patient compared with present-day        standard therapy.

In addition, the compounds according to the invention can also be usedin combination with radiotherapy and/or surgical intervention.

The present invention further provides medicaments which comprise atleast one compound of the invention, typically together with one or moreinert, non-toxic, pharmaceutically suitable excipients, and for the usethereof for the aforementioned purposes.

The compounds of the invention can act systemically and/or locally. Forthis purpose, they can be administered in a suitable manner, for exampleparenterally, possibly inhalatively or as implants or stents.

The compounds of the invention can be administered in administrationforms suitable for these administration routes.

Parenteral administration can bypass an absorption step (for exampleintravenously, intraarterially, intracardially, intraspinally orintralumbally) or include an absorption (for example intramuscularly,subcutaneously, intracutaneously, percutaneously or intraperitoneally).Administration forms suitable for parenteral administration includepreparations for injection and infusion in the form of solutions,suspensions, emulsions or lyophilizates. Preference is given toparenteral administration, especially intravenous administration.

In general, it has been found to be advantageous in the case ofparenteral administration to administer amounts of about 0.1 to 20mg/kg, preferably about 0.3 to 7 mg/kg, of body weight to achieveeffective results.

It may nevertheless be necessary in some cases to deviate from thestated amounts, and specifically as a function of body weight, route ofadministration, individual response to the active ingredient, nature ofthe preparation and time at which or interval over which administrationtakes place. Thus in some cases it may be sufficient to manage with lessthan the abovementioned minimum amount, while in other cases the upperlimit mentioned must be exceeded. In the case of administration ofgreater amounts, it may be advisable to divide them into severalindividual doses over the day.

EXAMPLES

The examples which follow illustrate the invention. The invention is notrestricted to these examples.

Unless stated otherwise, the percentages in the tests and examples whichfollow are percentages by weight; parts are parts by weight. Solventratios, dilution ratios and concentration data for the liquid/liquidsolutions are based in each case on volume.

Synthesis Routes:

By way of example for the working examples, the following schemes showillustrative synthesis routes leading to the working examples:

In the above reaction scheme, X₁, X₂, X₃, n and AK₂ have the meaningsgiven in formula (I).

a): HATU, DMF, N,N-diisopropylethylamine, RT; b) H₂, 10% Pd—C, methanol1.5 h, RT; c)1,1′-[(1,5-dioxopentane-1,5-diyl)bis(oxy)]dipyrrolidine-2,5-dione,N,N-diisopropylethylamine, DMF, stirring at RT overnight; d) AK2 in PBS,under argon addition of 3-5 equiv. of active ester dissolved in DMSO,stirring at RT under argon for 60 min, addition of another 3-5 equiv. ofactive ester dissolved in DMSO, stirring at RT under argon for 60 min,then purification by means of PD 10 columns equilibrated with PBS buffer(pH 7.2) (Sephadex® G-25, GE Healthcare) and subsequent concentration bymeans of ultracentrifugation and setting of the concentration desiredwith PBS buffer (pH 7.2)]. In the case of in vivo batches, this isoptionally followed by sterile filtration.

In the above reaction scheme, X₁, X₂, X₃, n and AK₁ have the meaningsgiven in formula (I).

a): HATU, DMF, N,N-diisopropylethylamine, RT; b) zinc chloride,trifluoroethanol, 50° C., EDTA c): HATU, DMF, N,N-diisopropylethylamine,RT; d) H₂, 10% Pd—C, methanol 1.5 h, RT; e)1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-1H-pyrrole-2,5-dione,N,N-diisopropylethylamine, DMF, stirring at RT; f) AK₁ dissolved in PBS,under argon addition of 3-4 equivalents of TCEP in PBS buffer and about30 min stirring at RT, then addition of 5-10 equivalents of compound Edissolved in DMSO, about 90 min of stirring at RT, then purification bymeans of PD 10 columns equilibrated with PBS buffer (pH 7.2) (Sephadex®G-25, GE Healthcare) and subsequent concentration by means ofultracentrifugation and setting of the concentration desired with PBSbuffer (pH 7.2)]. In the case of in vivo batches, this is optionallyfollowed by sterile filtration.

In the above reaction scheme, X₁, X₂, X₃, n and AK₁ have the meaningsgiven in formula (I).

a): HATU, DMF, N,N-diisopropylethylamine, RT; b) zinc chloride,trifluoroethanol, 50° C., EDTA c): HATU, DMF, N,N-diisopropylethylamine,RT; d) H₂, 10% Pd—C, methanol 1.5 h, RT; e)1-{2-[(2,5-dioxopyrrolidin-1-yl)oxy]-2-oxoethyl}-1H-pyrrole-2,5-dione,N,N-diisopropylethylamine, DMF, stirring at RT; f) AK1 dissolved in PBS,under argon addition of 3-4 equivalents of TCEP in PBS buffer and about30 min stirring at RT, then addition of 5-10 equivalents of compound Edissolved in DMSO, about 90 min of stirring at RT, then rebuffering topH 8 by means of PD 10 columns equilibrated with PBS buffer (pH 8)(Sephadex® G-25, GE Healthcare), then stirring at RT overnight, thenoptionally purification by means of PD 10 columns equilibrated with PBSbuffer (pH 7.2) (Sephadex® G-25, GE Healthcare) and subsequentconcentration by means of ultracentrifugation and setting of theconcentration desired with PBS buffer (pH 7.2)]. In the case of in vivobatches, this is optionally followed by sterile filtration.

A. Examples

Abbreviations and Acronyms:

-   ABCB1 ATP-binding cassette sub-family B member 1 (synonym for P-gp    and MDR1)-   abs. absolute-   Ac acetyl-   ACN acetonitrile-   aq. aqueous, aqueous solution-   ATP adenosine triphosphate-   BCRP breast cancer resistance protein, an efflux transporter-   BEP 2-bromo-1-ethylpyridinium tetrafluoroborate-   Boc tert-butoxycarbonyl-   br. broad (in NMR)-   Ex. Example-   BxPC3 human tumour cell line-   C concentration-   ca. circa, about-   CI chemical ionization (in MS)-   DAR drug-to-antibody ratio-   d doublet (in NMR)-   d day(s)-   TLC thin layer chromatography-   DCI direct chemical ionization (in MS)-   DCM dichloromethane-   dd doublet of doublets (in NMR)-   DMAP 4-N,N-dimethylaminopyridine-   DME 1,2-dimethoxyethane-   DMEM Dulbecco's Modified Eagle Medium (standardized nutrient medium    for cell culture)-   DMF N,N-dimethylformamide-   DMSO dimethyl sulfoxide-   D/P dye (fluorescent dye)/protein ratio-   DPBS, D-PBS, Dulbecco's phosphate-buffered salt solution-   DSMZ Deutsche Sammlung von Mikroorganismen and Zellkulturen (German    Collection of Microorganisms and Cell Cultures)-   PBS PBS=DPBS=D-PBS, pH 7.4, from Sigma, No D8537    -   Composition:    -   0.2 g KCl    -   0.2 g KH₂PO₄ (anhyd)    -   8.0 g NaCl    -   1.15 g Na₂HPO₄ (anhyd)    -   made up ad 1 l with H₂O-   dt doublet of triplets (in NMR)-   DTT DL-dithiothreitol-   EDC N′-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride-   EGFR epidermal growth factor receptor-   EI electron impact ionization (in MS)-   ELISA enzyme-linked immunosorbent assay-   eq. equivalent(s)-   ESI electrospray ionization (in MS)-   ESI-MicroTofq ESI-MicroTofq (name of the mass spectrometer with    Tof=time of flight and q=quadrupole)-   FCS foetal calf serum-   Fmoc (9H-fluoren-9-ylmethoxy)carbonyl-   sat. saturated-   GTP guanosine-5′-triphosphate-   H hour(s)-   HATU O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium    hexafluorophosphate-   HEPES 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid-   HOAc acetic acid-   HOAt 1-hydroxy-7-azabenzotriazole-   HOBt 1-hydroxy-1H-benzotriazole hydrate-   HOSu N-hydroxysuccinimide-   HPLC high-pressure, high-performance liquid chromatography-   IC₅₀ half-maximal inhibitory concentration-   i.m. intramuscularly, administration into the muscle-   i.v. intravenously, administration into the vein-   conc. concentrated-   KPL-4 human tumour cell line-   KU-19-19 human tumour cell line-   LC-MS liquid chromatography-coupled mass spectrometry-   LLC-PK1 cells Lewis lung carcinoma pork kidney cell line-   L-MDR human MDR1 transfected LLC-PK1 cells-   LoVo human tumour cell line-   M multiplet (in NMR)-   Me methyl-   MDR1 Multidrug resistance protein 1-   MeCN acetonitrile-   min minute(s)-   MOLM-13 human tumour cell line-   MS mass spectrometry-   MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide-   MV-4-11 human tumour cell line-   NB4 human tumour cell line-   NCI-H292 human tumour cell line-   NMM N-methylmorpholine-   NMP N-methyl-2-pyrrolidinone-   NMR nuclear magnetic resonance spectrometry-   NMRI mouse strain originating from the Naval Medical Research    Institute (NMRI)-   Nude mice experimental animals-   NSCLC non small cell lung cancer-   PBS phosphate-buffered salt solution-   Pd/C palladium on activated charcoal-   P-gp P-glycoprotein, a transporter protein-   PNGaseF enzyme for cleaving sugar-   quant. quantitative (in yield)-   quart quartet (in NMR)-   quint quintet (in NMR)-   Rec-1 human tumour cell line-   R_(f) retention index (in TLC)-   RT room temperature-   R_(t) retention time (in HPLC)-   s singlet (in NMR)-   s.c. subcutaneously, administration under the skin-   SCID mice test mice with severe combined immunodeficiency-   SK-HEP-1 human tumour cell line-   t triplet (in NMR)-   TBAF tetra-n-butylammonium fluoride-   TCEP tris(2-carboxyethyl)phosphine-   TEMPO (2,2,6,6-tetramethylpiperidin-1-yl)oxyl-   Teoc trimethylsilylethoxycarbonyl-   tert tertiary-   TFA trifluoroacetic acid-   THF tetrahydrofuran-   T3P® 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphinane 2,4,6-trioxide-   U251 human tumour cell line-   UV ultraviolet spectrometry-   v/v volume to volume ratio (of a solution)-   Z benzyloxycarbonyl

HPLC and LC-MS Methods:

Method 1 (LC-MS):

Instrument: Waters ACQUITY SQD UPLC System; column: Waters Acquity UPLCHSS T3 1.8μ50×1 mm; mobile phase A: 1 l of water+0.25 ml of 99% strengthformic acid; mobile phase B: 1 l of acetonitrile+0.25 ml of 99% strengthformic acid; gradient: 0.0 min 90% A→1.2 min 5% A→2.0 min 5% A; oven:50° C.; flow rate: 0.40 ml/min; UV detection: 208-400 nm.

Method 2 (LC-MS):

MS instrument type: Waters Synapt G2S; UPLC instrument type: WatersAcquity I-CLASS; column: Waters, BEH300, 2.1×150 mm, C18 1.7 μm; mobilephase A: 1 l of water+0.01% formic acid; mobile phase B: 1 l ofacetonitrile+0.01% formic acid; gradient: 0.0 min 2% B→1.5 min 2% B→8.5min 95% B→10.0 min 95% B; oven: 50° C.; flow rate: 0.50 ml/min; UVdetection: 220 nm

Method 3 (LC-MS):

MS instrument: Waters (Micromass) QM; HPLC instrument: Agilent 1100series; column: Agilent ZORBAX Extend-C18 3.0×50 mm 3.5 micron; mobilephase A: 1 l of water+0.01 mol of ammonium carbonate, mobile phase B: 1l of acetonitrile; gradient: 0.0 min 98% A→0.2 min 98% A→3.0 min 5%A→4.5 min 5% A ; oven: 40° C.; flow rate: 1.75 ml/min; UV detection: 210nm

Method 4 (LC-MS):

MS instrument type: Waters Synapt G2S; UPLC instrument type: WatersAcquity I-CLASS; column: Waters, HSST3, 2.1×50 mm, C18 1.8 μm; mobilephase A: 1 l of water+0.01% formic acid; mobile phase B: 1 l ofacetonitrile+0.01% formic acid; gradient: 0.0 min 10% B→0.3 min 10%B→1.7 min 95% B→2.5 min 95% B; oven: 50° C.; flow rate: 1.20 ml/min; UVdetection: 210 nm

Method 5 (LC-MS):

Instrument: Waters ACQUITY SQD UPLC System; column: Waters Acquity UPLCHSS T3 1.8μ50×1 mm; mobile phase A: 1 l of water+0.25 ml of 99% strengthformic acid; mobile phase B: 1 l of acetonitrile+0.25 ml of 99% strengthformic acid; gradient: 0.0 min 95% A→6.0 min 5% A→7.5 min 5% A; oven:50° C.; flow rate: 0.35 ml/min; UV detection: 210-400 nm.

Method 6 (LC-MS):

Instrument: Micromass Quattro Premier with Waters UPLC Acquity; column:Thermo Hypersil GOLD 1.9μ, 50×1 mm; mobile phase A: 1 l of water+0.5 mlof 50% strength formic acid; mobile phase B: 1 l of acetonitrile+0.5 mlof 50% strength formic acid; gradient: 0.0 min 97% A→0.5 min 97% A→3.2min 5% A→4.0 min 5% A oven: 50° C.; flow rate: 0.3 ml/min; UV detection:210 nm.

Method 7 (LC-MS):

Instrument: Agilent MS Quad 6150; HPLC: Agilent 1290; column: WatersAcquity UPLC HSS T3 1.8μ50×2.1 mm; mobile phase A: 1 l of water+0.25 mlof 99% strength formic acid; mobile phase B: 1 l of acetonitrile+0.25 mlof 99% strength formic acid; gradient: 0.0 min 90% A→0.3 min 90% A→1.7min 5% A→3.0 min 5% A oven: 50° C.; flow rate: 1.20 ml/min; UVdetection: 205-305 nm.

Method 8 (LC-MS):

MS instrument type: Waters Synapt G2S; UPLC instrument type: WatersAcquity I-CLASS; column: Waters, HSST3, 2.1×50 mm, C18 1.8 μm; mobilephase A: 1 l of water+0.01% formic acid; mobile phase B: 1 l ofacetonitrile+0.01% formic acid; gradient: 0.0 min 2% B→2.0 min 2% B→13.0min 90% B→15.0 min 90% B; oven: 50° C.; flow rate: 1.20 ml/min; UVdetection: 210 nm.

Method 9: (LC-MS Prep. Purification Method)

MS instrument: Waters, HPLC instrument: Waters (column Waters X-BridgeC18, 19 mm×50 mm, 5 μm, eluent A: water+0.05% ammonia, mobile phase B:acetonitrile (ULC) with gradient; flow rate: 40 ml/min; UV detection:DAD; 210-400 nm).

or

MS instrument: Waters, HPLC instrument: Waters (column Phenomenex Luna5μ C18(2) 100 A, AXIA Tech. 50×21.2 mm, eluent A: water+0.05% formicacid, eluent B: acetonitrile (ULC) with gradient; flow rate: 40 ml/min;UV detection: DAD; 210-400 nm).

Method 10: (LC-MS Analysis Method)

MS instrument: Waters SQD; HPLC instrument: Waters UPLC; column: ZorbaxSB-Aq (Agilent), 50 mm×2.1 mm, 1.8 μm; mobile phase A: water+0.025%formic acid, eluent B: acetonitrile (ULC)+0.025% formic acid; gradient:0.0 min 98% A−0.9 min 25% A−1.0 min 5% A−1.4 min 5% A−1.41 min 98% A−1.5min 98% A; oven: 40° C.; flow rate: 0.600 ml/min; UV detection: DAD; 210nm.

Method 11 (HPLC):

-   Instrument: HP1100 Series-   Column: Merck Chromolith SpeedROD RP-18e, 50-4.6 mm, Cat.    No.1.51450.0001, precolumn Chromolith Guard Cartridge Kit, RP-18e,    5-4.6mm, Cat. No. 1.51470.0001-   Gradient: flow rate 5 ml/min    -   injection volume 5 μl    -   solvent A: HClO4 (70%) in water (4 ml/l)    -   solvent B: acetonitrile    -   start 20% B    -   0.50 min 20% B    -   3.00 min 90% B    -   3.50 min 90% B    -   3.51 min 20% B    -   4.00 min 20% B    -   Column temperature: 40° C.-   Wavelength: 210 nm

Method 12 (LC-MS):

MS instrument type: Thermo Scientific FT-MS; instrument type UHPLC+:Thermo Scientific UltiMate 3000; column: Waters, HSST3, 2.1×75 mm, C181.8 μm; mobile phase A: 1 l of water+0.01% formic acid; mobile phase B:1 l of acetonitrile+0.01% formic acid; gradient: 0.0 min 10% B→2.5 min95% B→3.5 min 95% B; oven: 50° C.; flow rate: 0.90 ml/min; UV detection:210 nm/optimum integration path 210-300 nm.

Method 13: (LC-MS):

MS instrument: Waters (Micromass) Quattro Micro; instrument Waters UPLCAcquity; column: Waters BEH C18 1.7μ50×2.1 mm; mobile phase A: 1 l ofwater+0.01 mol of ammonium formate, mobile phase B: 1 l of acetonitrile;gradient: 0.0 min 95% A→0.1 min 95% A→2.0 min 15% A→2.5 min 15% A→2.51min 10% A→3.0 min 10% A; oven: 40° C.; flow rate: 0.5 ml/min; UVdetection: 210 nm.

Method 14: (LC-MS) (MCW-LTQ-POROSHELL-TFA98-10 min)

MS instrument type: ThermoFisherScientific LTQ-Orbitrap-XL; HPLCinstrument type: Agilent 1200SL; column: Agilent, POROSHELL 120, 3×150mm, SB—C18 2.7 μm; mobile phase A: 1 l of water+0.1% trifluoroaceticacid; mobile phase B: 1 l of acetonitrile+0.1% trifluoroacetic acid;gradient: 0.0 min 2% B→0.3 min 2% B→5.0 min 95% B 10.0 min 95% B; oven:40° C.; flow rate: 0.75 ml/min; UV detection: 210 nm

All reactants or reagents whose preparation is not described explicitlyhereinafter were purchased commercially from generally accessiblesources. For all other reactants or reagents whose preparation likewiseis not described hereinafter and which were not commercially obtainableor were obtained from sources which are not generally accessible, areference is given to the published literature in which theirpreparation is described.

Starting Compounds and Intermediates:

Intermediate C52

(1R)-1-[1-Benzyl-4-(2,5-difluorophenyl)-1H-pyrol-2-yl]-2,2-dimethylpropan-1-amine

10.00 g (49.01 mmol) of methyl 4-bromo-1H-pyrrole-2-carboxylate wereinitially charged in 100.0 ml of DMF, and 20.76 g (63.72 mmol) ofcaesium carbonate and 9.22 g (53.91 mmol) of benzyl bromide were added.The reaction mixture was stirred at RT overnight. The reaction mixturewas partitioned between water and ethyl acetate and the aqueous phasewas extracted with ethyl acetate. The combined organic phases were driedover magnesium sulfate and the solvent was evaporated under reducedpressure. The reaction was repreated with 90.0 g of methyl4-bromo-1H-pyrrole-2-carboxylate. The two combined reactions werepurified by preparative RP-HPLC (column: Daiso 300×100; 10μ, flow rate:250 ml/min, MeCN/water). The solvents were evaporated under reducedpressure and the residue was dried under high vacuum. This gave 125.15 g(87% of theory) of the compound methyl1-benzyl-4-bromo-1H-pyrrole-2-carboxylate.

LC-MS (Method 1): R_(t)=1.18 min; MS (ESIpos): m/z=295 [M+H]⁺.

Under argon, 4.80 g (16.32 mmol) of methyl1-benzyl-4-bromo-1H-pyrrole-2-carboxylate were initially charged in DMF,and 3.61 g (22.85 mmol) of (2,5-difluorophenyl)boronic acid, 19.20 ml ofsaturated sodium carbonate solution and 1.33 g (1.63 mmol) of[1,1′-bis(diphenylphosphino)ferrocene]-dichloropalladium(II):dichloromethanewere added. The reaction mixture was stirred at 85° C. overnight. Thereaction mixture was filtered through Celite and the filter cake waswashed with ethyl acetate. The organic phase was extracted with waterand then washed with saturated NaCl solution. The organic phase wasdried over magnesium sulfate and the solvent was evaporated underreduced pressure. The residue was purified on silica gel (mobile phase:cyclohexane/ethyl acetate 100:3). The solvents were evaporated underreduced pressure and the residue was dried under high vacuum. This gave3.60 g (67% of theory) of the compound methyl1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrole-2-carboxylate.

LC-MS (Method 7): R_(t)=1.59 min; MS (ESIpos): m/z=328 [M+H]⁺.

3.60 g (11.00 mmol) of methyl1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrole-2-carboxylate were initiallycharged in 90.0 ml of THF, and 1.04 g (27.50 mmol) of lithium aluminiumhydride (2.4 M in THF) were added at 0° C. The reaction mixture wasstirred at 0° C. for 30 minutes. Saturated potassium sodium tartratesolution was added at 0° C. and the reaction mixture was admixed withethyl acetate. The organic phase was extracted three times withsaturated potassium sodium tartrate solution. The organic phase waswashed once with saturated NaCl solution and dried over magnesiumsulfate. The solvent was evaporated under reduced pressure and theresidue was dissolved in 30.0 ml of dichloromethane. 3.38 g (32.99 mmol)of manganese(IV) oxide were added, and the mixture was stirred at RT for48 h. Another 2.20 g (21.47 mmol) of manganese(IV) oxide were added, andthe mixture was stirred at RT overnight. The reaction mixture wasfiltered through Celite and the filter cake was washed withdichloromethane. The solvent was evaporated under reduced pressure andthe residue 2.80 g of(1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrole-2-carbaldehyde) was usedwithout further purification in the next step of the synthesis.

LC-MS (Method 7): R_(t)=1.48 min; MS (ESIpos): m/z=298 [M+H]⁺.

28.21 g (94.88 mmol) of1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrole-2-carbaldehyde together with23.00 g (189.77 mmol) of (R)-2-methylpropane-2-sulfinamide wereinitially charged in 403.0 ml of absolute THF, and 67.42 g (237.21 mmol)of titanium(IV) isopropoxide were added and the mixture was stirred atRT overnight. 500 ml of saturated NaCl solution and 1000.0 ml of ethylacetate were added, and the mixture was stirred at RT for 1 h. Themixture was filtered through kieselguhr and the filtrate was washedtwice with saturated NaCl solution. The organic phase was dried overmagnesium sulfate, the solvent was evaporated under reduced pressure andthe residue was purified using Biotage Isolera (silica gel, column1500+340 g SNAP, flow rate 200 ml/min, ethyl acetate/cyclohexane 1:10).

LC-MS (Method 7): R_(t)=1.63 min; MS (ESIpos): m/z=401 [M+H]⁺.

25.00 g (62.42 mmol) of(R)—N-{(E/Z)-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]methylene}-2-methylpropane-2-sulfinamidewere initially charged in absolute THF under argon and cooled to −78° C.12.00 g (187.27 mmol) of tert-butyllithium (1.7 M solution in pentane)were then added at −78° C. and the mixture was stirred at thistemperature for 3 h. At −78° C., 71.4 ml of methanol and 214.3 ml ofsaturated ammonium chloride solution were then added successively andthe reaction mixture was allowed to warm to RT and stirred at RT for 1h. The mixture was diluted with ethyl acetate and washed with water. Theorganic phase was dried over magnesium sulfate and the solvent wasevaporated under reduced pressure. The residue(R)—N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2-methylpropane-2-sulfinamidewas used without further purification in the next step of the synthesis.

LC-MS (Method 6): R_(t)=2.97 min; MS (ESIpos): m/z=459 [M+H]⁺.

28.00 g (61.05 mmol) of(R)—N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2-methylpropane-2-sulfinamidewere initially charged in 186.7 ml of 1,4-dioxane, and 45.8 ml of HCl in1,4-dioxane solution (4.0 M) were then added. The reaction mixture wasstirred at RT for 2 h and the solvent was evaporated under reducedpressure.

The residue was purified by preparative HPLC (column: Kinetix 100×30;flow rate: 60 ml/min, MeCN/water). The acetonitrile was evaporated underreduced pressure and dichloromethane was added to the aqueous residue.The organic phase was washed with sodium bicarbonate solution and driedover magnesium sulfate. The solvent was evaporated under reducedpressure and the residue was dried under high vacuum. This gave 16.2 g(75% of theory) of the title compound.

LC-MS (Method 6): R_(t)=2.10 min; MS (ESIpos): m/z=338 [M−NH₂]⁺, 709[2M₊H]⁺.

¹H-NMR (400 MHz, DMSO-d₆): δ [ppm]=0.87 (s, 9H), 1.53 (s, 2H), 3.59 (s,1H), 5.24 (d, 2H), 6.56 (s, 1H), 6.94 (m, 1H), 7.10 (d, 2H), 7.20 (m,1H), 7.26 (m, 2H), 7.34 (m, 2H), 7.46 (m, 1H).

Intermediate C58

(2S)-4-[{(1R)-1-[1-Benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butanoicacid

4.3 g (12.2 mmol) of Intermediate C52 were dissolved in 525 ml of DCM,and 3.63 g (17.12 mmol) of sodium triacetoxyborohydride and 8.4 ml ofacetic acid were added. After 5 min of stirring at RT, 8.99 g (24.5mmol) of Intermediate L57 dissolved in 175 ml of DCM were added and thereaction was stirred at RT for a further 45 min. The reaction was thendiluted with 300 ml of DCM and washed twice with 100 ml of sodiumbicarbonate solution and once with saturated NaCl solution. The organicphase was dried over magnesium sulfate, the solvent was evaporated underreduced pressure and the residue was dried under high vacuum. Theresidue was then purified by preparative RP-HPLC (column: ChromatorexC18). After combination of the appropriate fractions, the solvent wasevaporated under reduced pressure and the residue was dried under highvacuum. This gave 4.6 g (61% of theory) of methyl(2S)-4-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino)-2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butanoate.

LC-MS (Method 12): R_(t)=1.97 min; MS (ESIpos): m/z=614 (M+H)⁺.

2.06 g (3.36 mmol) of this intermediate were initially charged in 76 mlof DCM and acylated with 0.81 ml (7.17 mmol) of 2-chloro-2-oxoethylacetate in the presence of 2.1 ml of triethylamine. After 20 h ofstirring at RT, 0.36 ml of 2-chlor-2-oxoethyl acetate and 0.94 ml oftriethylamine were added and the reaction was stirred at RT for afurther 15 min. The mixture was then diluted with 500 ml of ethylacetate and extracted successively twice with 300 ml of 5% citric acid,twice with 300 ml of saturated sodium hydrogencarbonate solution andonce with 100 ml of saturated sodium chloride solution and then driedover magnesium sulfate and concentrated. Drying under high vacuum gave2.17 g (79% of theory) of the protected intermediate.

LC-MS (Method 1): R_(t)=1.48 min; MS (ESIpos): m/z=714 (M+H)⁺.

2.17 mg (2.64 mmol) of this intermediate were dissolved in 54 ml of THFand 27 ml of water, and 26 ml of a 2-molar lithium hydroxide solutionwere added. The mixture was stirred at RT for 30 min and then adjustedto a pH between 3 and 4 using 1.4 ml of TFA. The mixture wasconcentrated under reduced pressure. Once most of the THF had beendistilled off, the aqueous solution was extracted twice with DCM andthen concentrated to dryness under reduced pressure. The residue waspurified by preparative HPLC (column: Chromatorex C18). Aftercombination of the appropriate fractions, the solvent was evaporatedunder reduced pressure and the residue was lyophilized fromacetonitrile/water. This gave 1.1 g (63% of theory) of the titlecompound.

LC-MS (Method 1): R_(t)=1.34 min; MS (ESIpos): m/z=656 (M−H)⁻.

¹H-NMR (400 MHz, DMSO-d₆): δ [ppm]=0.03 (s, 9H), 0.58 (m, 1H), 0.74-0.92(m, 11H), 1.40 (m, 1H), 3.3 (m, 2H), 3.7 (m, 1H), 3.8-4.0 (m, 2H), 4.15(q, 2H), 4.9 and 5.2 (2d, 2H), 5.61 (s, 1H), 6.94 (m, 2H), 7.13-7.38 (m,7H), 7.48 (s, 1H), 7.60 (m, 1H), 12.35 (s, 1H).

Intermediate C61

N-[(2S)-4-[{(1R)-1-[1-Benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butanoyl]-beta-alanine

The title compound was prepared by coupling 60 mg (0.091 mmol) ofIntermediate C58 with methyl ß-alaninate, followed by ester cleavagewith 2M lithium hydroxide solution. This gave 67 mg (61% of theory) ofthe title compound over 2 steps.

LC-MS (Method 1): R_(t)=1.29 min; MS (ESIpos): m/z=729 (M+H)⁺.

Intermediate C102

(2S)-4-[{(1R)-1-[1-Benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-2-{[(benzyloxy)carbonyl]amino}butanoicacid

First, intermediate C52 was reductively alkylated with benzyl(2S)-2-{[(benzyloxy)carbonyl]amino}-4-oxobutanoate analogously toIntermediate C2. The secondary amino group was then acylated with2-chloro-2-oxoethyl acetate, and the two ester groups were thenhydrolysed with 2M lithium hydroxide solution in methanol.

LC-MS (Method 1): R_(t)=1.31 min; MS (ESIpos): m/z=646 (M−H)⁻.

Intermediate C110(D)

DibenzylN-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butanoyl}-beta-alanyl-D-glutamate

The title compound was prepared by coupling dibenzyl D-glutamate, whichhad been released beforehand from its p-toluenesulfonic acid salt bypartitioning between ethyl acetate and 5% sodium hydrogencarbonatesolution, with Intermediate C61 in the presence of HATU andN,N-diisopropylethylamine and subsequent detachment of the Teocprotecting group by means of zinc chloride in trifluoroethanol.

LC-MS (Method 1): R_(t)=1.08 min; MS (ESIpos): m/z=894 [M+H]⁺.

Intermediate C111

Di-tert-butylN-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butanoyl}-beta-alanyl-D-glutamate

First of all, the dipeptide derivative di-tert-butylbeta-alanyl-D-glutamate was prepared by conventional methods of peptidechemistry by coupling of commercially availableN-[(benzyloxy)carbonyl]-beta-alanine and di-tert-butyl D-glutamatehydrochloride (1:1) in the presence of HATU and subsequenthydrogenolytic detachment of the Z protecting group. The title compoundwas then prepared by coupling this intermediate with Intermediate C102in the presence of HATU and N,N-diisopropylethylamine and subsequentdetachment of the Z protecting group by hydrogenation over 10% palladiumon activated carbon in DCM/m ethanol 1:1 at RT under standard hydrogenpressure for 1 hour.

LC-MS (Method 1): R_(t)=1.06 min; MS (ESIpos): m/z=826 [M+H]⁺.

Intermediate C117

Trifluoroacetic acid dibenzylN-{(2S)-2-(L-asparaginylamino)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butanoyl}-beta-alanyl-D-glutamatesalt

Intermediate C110D and2,5-dioxopyrrolidin-1-yl-N²-(tert-butoxycarbonyl)-L-asparaginate (251mg, 764 μmol) were dissolved in 21 ml of DMF andN,N-diisopropylethylamine (363 μl, 2.01 mmol) was added. The reactionwas stirred at RT and then purified directly by prep. RP-HPLC (column:Chromatorex C18-10). The solvents were evaporated under reduced pressureand the residue was lyophilized.

The intermediate obtained (578 mg, 52 μmol) was dissolved in 20.0 ml oftrifluoroethanol. The reaction mixture was admixed with zinc chloride(426 mg, 3.13 mmol) and stirred at 50° C. for 40 min. The mixture wasadmixed with ethylenediamine-N,N,N′,N′-tetraacetic acid (914 mg, 3.13mmol) and diluted with 20 ml of water, TFA (200 μl) was added and themixture was stirred briefly. The mixture was filtered and purified bypreparative RP-HPLC (column: Chromatorex C18-5; 125×40, flow rate: 100ml/min, MeCN/water, 0.1% TFA gradient). Lyophilization gave the titlecompound.

Intermediate L57

Methyl(2S)-4-oxo-2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butanoate

500.0 mg (2.72 mmol) of methyl L-asparaginate hydrochloride and 706.3 mg(2.72 mmol) of 2-(trimethylsilyl)ethyl2,5-dioxopyrrolidine-1-carboxylate were initially charged in 5.0 ml of1,4-dioxane, and 826.8 mg (8.17 mmol) of triethylamine were added. Thereaction mixture was stirred at RT overnight. The reaction mixture waspurified directly by preparative RP-HPLC (column: Reprosil 250×40; 10μ,flow rate 50 ml/min, MeCN/water, 0.1% TFA). The solvents were thenevaporated under reduced pressure and the residue was dried under highvacuum. This gave 583.9 mg (74% of theory) of the compound(3S)-4-methoxy-4-oxo-3-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butanoicacid.

LC-MS (Method 1): R_(t)=0.89 min; MS (ESIneg): m/z=290 (M−H)⁻.

592.9 mg of(3S)-4-methoxy-4-oxo-3-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butanoicacid were initially charged in 10.0 ml of 1,2-dimethoxyethane, themixture was cooled to −15° C. and 205.8 mg (2.04 mmol) of4-methylmorpholine and 277.9 mg (2.04 mmol) of isobutyl chloroformatewere added. The precipitate was filtered off with suction after 15 minand twice with in each case 10.0 ml of 1,2-dimethoxyethane. The filtratewas cooled to −10° C., and 115.5 mg (3.05 mmol) of sodium borohydridedissolved in 10 ml of water were added with vigorous stirring. Thephases were separated and the organic phase was washed once each withsaturated sodium hydrogencarbonate solution and saturated NaCl solution.The organic phase was dried over magnesium sulfate, the solvent wasevaporated under reduced pressure and the residue was dried under highvacuum. This gave 515.9 mg (91% of theory) of the compound methylN-{[2-(trimethylsilyl)ethoxy]carbonyl}-L-homoserinate.

LC-MS (Method 1): R_(t)=0.87 min; MS (ESIpos): m/z=278 (M+H)⁺.

554.9 mg (2.00 mmol) of methylN-{[2-(trimethylsilyl)ethoxy]carbonyl}-L-homoserinate were initiallycharged in 30.0 ml of dichloromethane, and 1.27 g (3.0 mmol) ofDess-Martin periodinane and 474.7 mg (6.00 mmol) of pyridine were added.The mixture was stirred at RT overnight. After 4 h, the reaction wasdiluted with dichloromethane and the organic phase was washed in eachcase three times with 10% strength Na₂S₂O₃ solution, 10% strength citricacid solution and saturated sodium hydrogencarbonate solution. Theorganic phase was dried over magnesium sulfate and the solvent wasevaporated under reduced pressure. This gave 565.7 mg (97% of theory) ofthe title compound.

¹H-NMR (400 MHz, DMSO-d₆): δ [ppm]=0.03 (s, 9H), 0.91 (m, 2H), 2.70-2.79(m, 1H), 2.88 (dd, 1H), 3.63 (s, 3H), 4.04 (m, 2H), 4.55 (m, 1H), 7.54(d, 1H), 9.60 (t, 1H).

Intermediate L95

N-[(Benzyloxy)carbonyl]-L-valyl-L-alanine

This intermediate was prepared proceeding fromN-[(benzyloxy)carbonyl]-L-valine and tert-butyl L-alaninatehydrochloride by conventional methods of peptide chemistry.

LC-MS (Method 12): R_(t)=1.34 min; MS (ESIpos): m/z=323.16 (M+H)⁺.

Intermediate L103

N-(Pyridin-4-ylacetyl)-L-alanyl-L-alanyl-L-asparagine trifluoroacetate

The title compound was prepared by conventional methods of peptidechemistry commencing with the coupling of 4-pyridineacetic acid withcommercially available tert-butyl L-alanyl-L-alaninate in the presenceof HATU and N,N-diisopropylethylamine, followed by deprotection withtrifluoroacetic acid, coupling to tert-butyl L-asparaginate andsubsequent deprotection of the carboxyl group with trifluoroacetic acid.

LC-MS (Method 1): R_(t)=0.15 min; MS (ESIpos): m/z=394 (M+H)⁺.

Intermediate L116

N-[(Benzyloxy)carbonyl]-L-alanyl-N-methyl-L-alanine

The title compound was prepared from commercially availableN-[(benzyloxy)carbonyl]-L-alanine by classical methods of peptidechemistry via coupling with tert-butyl N-methyl-L-alaninatehydrochloride salt in the presence of HATU and finally by removal of thetert-butyl ester protective group with TFA.

LC-MS (Method 1): R_(t)=0.68 min; MS (ESIpos): m/z=309 [M+H]⁺

Intermediate L117

N-[(Benzyloxy)carbonyl]-L-alanyl-N-methyl-L-alanyl-L-asparaginetrifluoroacetic acid salt

The title compound was prepared from commercially available 4-tert-butylL-asparaginate by classical methods of peptide chemistry via couplingwith N-[(benzyloxy)carbonyl]-L-alanyl-N-methyl-L-alanine (IntermediateL116) in the presence of HATU and finally by removal of the tert-butylester protective group with TFA.

LC-MS (Method 1): R_(t)=0.57 min; MS (ESIneg): m/z=421 [M−H]⁻

Intermediate L118

N-[(Benzyloxy)carbonyl]-L-alanyl-N-methyl-L-alanyl-L-alanine

The title compound was prepared from commercially available tert-butylL-alaninate hydrochloride salt by classical methods of peptide chemistryvia coupling with N-[(benzyloxy)carbonyl]-L-alanyl-N-methyl-L-alanine(Intermediate L116) in the presence of HATU and finally by removal ofthe tert-butyl ester protective group with TFA.

LC-MS (Method 12): R_(t)=1.25 min; MS (ESIneg): m/z=378 [M−H]⁻

Intermediate L121

N-[(Benzyloxy)carbonyl]-L-alanyl-N-methyl-L-alanyl-L-leucine

The title compound was prepared from commercially available tert-butylL-leucinate hydrochloride salt by classical methods of peptide chemistryvia coupling with N-[(benzyloxy)carbonyl]-L-alanyl-N-methyl-L-alanine(Intermediate L116) in the presence of HATU and finally by removal ofthe tert-butyl ester protective group with TFA.

LC-MS (Method 12): R_(t)=0.83 min; MS (ESIneg): m/z=420 [M−H]⁻

Intermediate L122

(5S,8S,11S)-11-(2-Amino-2-oxoethyl)-8-[2-(benzyloxy)-2-oxoethyl]-5-methyl-3,6,9-trioxo-1-phenyl-2-oxa-4,7,10-triazadodecan-12-oicacid

The title compound was prepared from commercially available4-benzyl-1-tert-butyl-L-aspartate hydrochloride (1:1) by classicalmethods of peptide chemistry via initially coupling with2,5-dioxopyrrolidin-1-yl-N-[(benzyloxy)carbonyl]-L-alaninate, thenremoving the tert-butyl ester protective group with TFA, then subsequentcoupling with 4-tert-butyl L-asparaginate in the presence of HATU andfinally by once more removing the tert-butyl ester protective group withTFA.

LC-MS (Method 1): R_(t)=0.76 min; MS (ESIpos): m/z=543 [M+H]+

Intermediate L138

1-Bromo-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecan-18-oic acid

The title compound was prepared by coupling of1-amino-3,6,9,12-tetraoxapentadecan-15-oic acid with bromoaceticanhydride in the presence of N,N-diisopropylethylamine.

LC-MS (Method 5): R_(t)=1.05 min; MS (ESIpos): m/z=386 and 388 (M+H)⁺.

Intermediate Q1

N-[(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-L-alanyl-N-methyl-L-alanyl-N¹-{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-1-[(3-{[(1R)-1,3-dicarboxypropyl]amino}-3-oxopropyl)amino]-1-oxobutan-2-yl}-L-aspartamide

The title compound was prepared proceeding from compound C110D, first bycoupling to Intermediate L117 in the presence of HATU andN,N-diisopropylethylamine. In the next step, all protecting groups wereremoved by hydrogenation over 10% palladium on activated carbon inDCM-methanol 1:1 under standard hydrogen pressure at RT for 1 hour andthe deprotected intermediate was then converted to the title compound byreaction with1-{2-[(2,5-dioxopyrrolidin-1-yl)oxy]-2-oxoethyl}-1H-pyrrole-2,5-dione inthe presence of N,N-diisopropylethylamine.

LC-MS (Method 12): R_(t)=1.66 min; MS (ESIneg): m/z=1119 [M−H]⁻.

Intermediate Q2

N-{5-[(2,5-Dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}-L-alanyl-N-methyl-L-alanyl-N¹-{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-1-[(3-{[(1R)-1,3-dicarboxypropyl]amino}-3-oxopropyl)amino]-1-oxobutan-2-yl}-L-aspartamide

The title compound was prepared proceeding from compound C110D, first bycoupling to Intermediate L117 in the presence of HATU andN,N-diisopropylethylamine. In the next step, all protecting groups wereremoved by hydrogenation over 10% palladium on activated carbon inDCM-methanol 1:1 under standard hydrogen pressure at RT for 1 hour andthe deprotected intermediate was then converted to the title compound byreaction with1,1′-[(1,5-dioxopentane-1,5-diyl)bis(oxy)]dipyrrolidine-2,5-dione in thepresence of N,N-diisopropylethylamine.

LC-MS (Method 1): R_(t)=0.93 min; MS (ESIpos): m/z=1195 [M+H]⁺.

Intermediate Q3

N-{5-[(2,5-Dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}-L-alanyl-L-alanyl-N¹-{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-1-[(3-{[(1R)-1,3-dicarboxypropyl]amino}-3-oxopropyl)amino]-1-oxobutan-2-yl}-L-aspartamide

The title compound was prepared from Compound C117 first by coupling toN-(tert-butoxycarbonyl)-L-alanyl-L-alanine in the presence of HATU andN,N-diisopropylethylamine. The intermediate was then taken up intrifluoroethanol and the tert-butoxycarbonyl-protected amine wasreleased by stirring at 50° C. in the presence of zinc chloride. In thenext step, all benzyl protecting groups were removed by hydrogenationover 10% palladium on activated carbon in DCM-methanol 1:1 understandard hydrogen pressure at RT for 1 hour and the deprotectedintermediate was then converted to the title compound by reaction with1,1′-[(1,5-dioxopentane-1,5-diyl)bis(oxy)]dipyrrolidine-2,5-dione in thepresence of N,N-diisopropylethylamine.

LC-MS (Method 1): R_(t)=0.90 min; MS (ESIneg): m/z=1181 [M−H]⁻.

Intermediate Q4

N-[(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-alanyl-N-methyl-L-alanyl-N¹-{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-1-[(3-{[(1R)-1,3-dicarboxypropyl]amino}-3-oxopropyl)amino]-1-oxobutan-2-yl}-L-aspartamide

The title compound was prepared proceeding from compound C110D, first bycoupling to Intermediate L117 in the presence of HATU andN,N-diisopropylethylamine. In the next step, all protecting groups wereremoved by hydrogenation over 10% palladium on activated carbon inDCM-methanol 1:1 under standard hydrogen pressure at RT for 1 hour andthe deprotected intermediate was then converted to the title compound byreaction with1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-1H-pyrrole-2,5-dione inthe presence of N,N-diisopropylethylamine.

LC-MS (Method 1): R_(t)=3.2 min; MS (ESIpos): m/z=1177 [M+H]⁺.

Intermediate Q5

N-{5-[(2,5-Dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}-L-alanyl-N-methyl-L-alanyl-N-{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-1-[(3-{[(1R)-1,3-dicarboxypropyl]amino}-3-oxopropyl)amino]-1-oxobutan-2-yl}-L-alaninamide

The title compound was prepared proceeding from compound C110D, first bycoupling to Intermediate L118 in the presence of HATU andN,N-diisopropylethylamine. In the next step, all protecting groups wereremoved by hydrogenation over 10% palladium on activated carbon inDCM-methanol 1:1 under standard hydrogen pressure at RT for 1 hour andthe deprotected intermediate was then converted to the title compound byreaction with1,1′-[(1,5-dioxopentane-1,5-diyl)bis(oxy)]dipyrrolidine-2,5-dione in thepresence of N,N-diisopropylethylamine.

LC-MS (Method 1): R_(t)=0.96 min; MS (ESIpos): m/z=1152 [M+H]⁺.

Intermediate Q6

N-{(2S)-4-[{(1R)-1-[1-Benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-2-[(N-{5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}-L-valyl-L-alanyl)amino]butanoyl}-beta-alanyl-D-glutamicacid

The title compound was prepared proceeding from compound C110D, first bycoupling to Intermediate L95 in the presence of HATU andN,N-diisopropylethylamine. In the next step, all protecting groups wereremoved by hydrogenation over 10% palladium on activated carbon inDCM-methanol 1:1 under standard hydrogen pressure at RT for 1 hour andthe deprotected intermediate was then converted to the title compound byreaction with1,1′-[(1,5-dioxopentane-1,5-diyl)bis(oxy)]dipyrrolidine-2,5-dione in thepresence of N,N-diisopropylethylamine.

LC-MS (Method 1): R_(t)=0.98 min; MS (ESIpos): m/z=1095 [M+H]⁺.

Intermediate Q7

N-[(2S)-4-[{(1R)-1-[1-Benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-2-({N-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-L-valyl-L-alanyl}amino)butanoyl]-beta-alanyl-D-glutamicacid

The title compound was prepared proceeding from compound C110D, first bycoupling to Intermediate L95 in the presence of HATU andN,N-diisopropylethylamine. In the next step, all protecting groups wereremoved by hydrogenation over 10% palladium on activated carbon inDCM-methanol 1:1 under standard hydrogen pressure at RT for 1 hour andthe deprotected intermediate was then converted to the title compound byreaction with1-{2-[(2,5-dioxopyrrolidin-1-yl)oxy]-2-oxoethyl}-1H-pyrrole-2,5-dione inthe presence of N,N-diisopropylethylamine.

LC-MS (Method 1): R_(t)=0.98 min; MS (ESIpos): m/z=1021 [M+H]⁺.

Intermediate Q8

N-{5-[(2,5-Dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}-L-alanyl-N-methyl-L-alanyl-N¹-{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-1-[(3-{[(1R)-1,3-dicarboxypropyl]amino}-3-oxopropyl)amino]-1-oxobutan-2-yl}-L-leucinamide

The title compound was prepared proceeding from compound C110D, first bycoupling to Intermediate L121 in the presence of HATU andN,N-diisopropylethylamine. In the next step, all protecting groups wereremoved by hydrogenation over 10% palladium on activated carbon inethanol under standard hydrogen pressure at RT for 1 hour and thedeprotected intermediate was then converted to the title compound byreaction with1,1′-[(1,5-dioxopentane-1,5-diyl)bis(oxy)]dipyrrolidine-2,5-dione in thepresence of N,N-diisopropylethylamine.

LC-MS (Method 1): R_(t)=1.02 min; MS (ESIpos): m/z=1194 [M+H]⁺.

Intermediate Q9

N-{5-[(2,5-Dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}-L-alanyl-N-methyl-L-alpha-aspartyl-N¹-{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-1-[(3-{[(1R)-1,3-dicarboxypropyl]amino}-3-oxopropyl)amino]-1-oxobutan-2-yl}-L-aspartamide

The title compound was prepared proceeding from compound C110D, first bycoupling to Intermediate L122 in the presence of HATU andN,N-diisopropylethylamine. In the next step, all protecting groups wereremoved by hydrogenation over 10% palladium on activated carbon inmethanol under standard hydrogen pressure at RT for 1 hour and thedeprotected intermediate was then converted to the title compound byreaction with 3 equiv. of1,1′-[(1,5-dioxopentane-1,5-diyl)bis(oxy)]dipyrrolidine-2,5-dione in thepresence of N,N-diisopropylethylamine.

LC-MS (Method 1): R_(t)=0.89 min; MS (ESIpos): m/z=1225 [M+H]⁺.

Intermediate Q10

N-(Bromoacetyl)-L-alanyl-N-methyl-L-alanyl-N¹-{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-1-[(3-{[(1R)-1,3-dicarboxypropyl]amino}-3-oxopropyl)amino]-1-oxobutan-2-yl}-L-aspartamide

The title compound was prepared proceeding from compound C110D, first bycoupling to Intermediate L117 in the presence of HATU andN,N-diisopropylethylamine. In the next step, all protecting groups wereremoved by hydrogenation over 10% palladium on activated carbon inDCM-methanol 1:1 under standard hydrogen pressure at RT for 1 hour andthe deprotected intermediate was then converted to the title compound byreaction with bromoacetic anhydride in the presence of 3 equiv. ofN,N-diisopropylethylamine.

LC-MS (Method 1): R_(t)=0.95 min; MS (ESIpos): m/z=1104 and 1106 [M+H]⁺.

Intermediate Q11

N-(18-Bromo-17-oxo-4,7,10,13-tetraoxa-16-azaoctadecan-1-oyl)-L-alanyl-N-methyl-L-alanyl-N¹-{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-1-[(3-{[(1R)-1,3-dicarboxypropyl]amino}-3-oxopropyl)amino]-1-oxobutan-2-yl}-L-aspartamide

The synthesis of the title compound was carried out by initiallycoupling Intermediate C111 with intermediate L117 in DMF in the presenceof 1.5 equiv. HATU and 3 equiv. of N,N-diisopropylethylamine.Subsequently, the Z protective group was removed by a 2-hourhydrogenation over 10% palladium on activated carbon in ethanol underhydrogen standard pressure at RT. The deprotected intermediate was thenreacted with Intermediate L138 in DMF in the presence of 1.5 equiv. ofHATU and 3 equiv. of N,N-diisopropylethylamine. In the last step,cleavage of the tert-butyl ester groups by 2 h of stirring at 50° C.with 8 equivalents of zinc chloride in trifluoroethanol gave the titlecompound.

LC-MS (Method 8): R_(t)=4.06 min; MS (ESI-pos): m/z=1353 [M+H]⁺.

B: Preparation of Antibody-Drug Conjugates (ADC)

B-1. General method for Generation of Antibodies

The protein sequence (amino acid sequence) of the antibodies used, forexample TPP-981, TPP-1015, TPP-6013, TPP-7006, TPP-7007, TPP-8382,TPP-8987, TPP-8988, TPP-9476, TPP-9574 and TPP-9580, was transformedinto a DNA sequence that encodes the corresponding protein by a methodknown to those skilled in the art and inserted into an expression vectorsuitable for transient mammalian cell culture (as described by Tom etal., Chapter 12 in Methods Express: Expression Systems, edited byMichael R. Dyson and Yves Durocher, Scion Publishing Ltd, 2007).

B-2. General Method for Expression of Antibodies in Mammalian Cells

The antibodies, for example TPP-981, TPP-1015, TPP-6013, TPP-7006,TPP-7007, TPP-8382, TPP-8987, TPP-8988, TPP-9476, TPP-9574 and TPP-9580,were produced in transient mammalian cell cultures, as described by Tomet al., Chapter 12 in Methods Express: Expression Systems, edited byMichael R. Dyson and Yves Durocher, Scion Publishing Ltd, 2007.

B-3. General Method for Purification of Antibodies from CellSupernatants

The antibodies, for example TPP-981, TPP-1015, TPP-6013, TPP-7006,TPP-7007, TPP-8382, TPP-8987, TPP-8988, TPP-9476, TPP-9574 and TPP-9580,were obtained from the cell culture supernatants. The cell supernatantswere clarified by centrifugation of cells. The cell supernatant was thenpurified by affinity chromatography on a MabSelect Sure (GE Healthcare)chromatography column. To this end, the column was equilibrated in DPBSpH 7.4 (Sigma/Aldrich), the cell supernatant was applied and the columnwas washed with about 10 column volumes of DPBS pH 7.4+500 mM sodiumchloride. The antibodies were eluted in 50 mM sodium acetate pH 3.5+500mM sodium chloride and then purified further by gel filtrationchromatography on a Superdex 200 column (GE Healthcare) in DPBS pH 7.4.

Commercially available antibodies were purified by standardchromatography methods (protein A chromatography, preparative gelfiltration chromatography (SEC—size exclusion chromatography)) from thecommercial products.

B-4. General Method for Coupling to Cysteine Side Chains

The following antibodies were used in the coupling reactions:

Examples a: TPP-981 cetuximab (anti-EGFR AK)

Examples c: TPP-6013 (anti-CD123 AK)

-   -   TPP-8987 (anti-CD123 AK)    -   TPP-8988 (anti-CD123 AK)    -   TPP-9476 (anti-CD123 AK)

Examples h: TPP-8382 (anti-B7H3 AK)

-   -   Examples e: TPP-1015 (anti-Her2 AK)    -   Examples k: TPP-7006 (anti-TWEAKR AK) TPP-7007 (anti-TWEAKR AK)

Examples x: TPP-9574 (anti-CXCR5 AK)

-   -   TPP-9580 (anti-CXCR5 AK)

The coupling reactions were usually carried out under argon.

Between 2 and 5 equivalents of tris(2-carboxyethyl)phosphinehydrochloride (TCEP), dissolved in PBS buffer, were added to a solutionof the appropriate antibody in PBS buffer in the concentration rangebetween 1 mg/ml and 20 mg/ml, preferably in the range of about 10 mg/mlto 15 mg/ml, and the mixture was stirred at RT for 1 h. For thispurpose, the solution of the respective antibody used can be employed atthe concentrations stated in the working examples, or it may optionallyalso be diluted with PBS buffer to about half of the stated startingconcentration in order to get into the preferred concentration range.Subsequently, depending on the intended loading, from 2 to 12equivalents, preferably about 5-10 equivalents of the maleimideprecursor compound or halide precursor compound to be coupled were addedas a solution in DMSO. Here, the amount of DMSO should not exceed 10% ofthe total volume. The mixture was stirred in the case of maleimideprecursors for 60-240 min at RT and in the case of halide precursorsbetween 8 and 24 h at RT and then applied to PBS-equilibrated PD 10columns (Sephadex® G-25, GE Healthcare) and eluted with PBS buffer.Generally, unless indicated otherwise, 5 mg of the antibody in questionin PBS buffer were used for the reduction and the subsequent coupling.Purification on the PD10 column thus in each case afforded solutions ofthe respective ADCs in 3.5 ml PBS buffer. The sample was thenconcentrated by ultracentrifugation and optionally rediluted with PBSbuffer. If required, for better removal of low-molecular weightcomponents, concentration by ultrafiltration was repeated afterredilution with PBS buffer. For biological tests, if required, theconcentrations of the final ADC samples were optionally adjusted to therange of 0.5-15 mg/ml by redilution. The respective proteinconcentrations, stated in the working examples, of the ADC solutionswere determined. Furthermore, antibody loading (drug/mAb ratio) wasdetermined using the methods described under B-6.

Depending on the linker, the ADCs shown in the examples may also bepresent to a lesser or higher degree in the form of the hydrolysedopen-chain succinamides linked to the antibodies.

Particularly the KSP-I-ADCs linked via the linker substructure

to thiol groups of the antibodies can optionally also be preparedselectively by rebuffering after the coupling and stirring at pH 8 forabout 20-24 h according to Scheme 28 in the ADCs linked via open-chainsuccinamides.

#1 represents the sulfur bridge to the antibody, and #2 the point ofattachment to the modified KSP inhibitor

Such ADCs where the linker is attached to the antibodies throughhydrolysed open-chain succinamides can optionally also be preparedselectively by the small scale and large scale couplings shown here byway of example:

Between 2 and 7 equivalents of tris(2-carboxyethyl)phosphinehydrochloride (TCEP), dissolved in PBS buffer, were added to a solutionof 2-5 mg of the appropriate antibody in PBS buffer in the concentrationrange between 1 mg/ml and 20 mg/ml, preferably in the range of about 5mg/ml to 15 mg/ml, and the mixture was stirred at RT for 30 min to 1 h.Subsequently, depending on the intended loading, from 2 to 20equivalents, preferably about 5-10 equivalents of the maleimideprecursor compound to be coupled were added as a solution in DMSO. Toachieve higher DARs, it is also possible to use 15-20 equivalents. Here,the amount of DMSO should not exceed 10% of the total volume. Themixture was stirred at RT for 60-240 min. The eluate was diluted withPBS buffer pH 8 to a concentration of 1-5 mg/ml and then passed througha PD 10 column (Sephadex® G-25, GE Healthcare) equilibrated with PBSbuffer pH 8, and eluted with PBS buffer pH 8. The eluate was stirred atRT under argon overnight. Subsequently, the solution was concentrated byultracentrifugation and rediluted with PBS buffer (pH 7.2).

Medium-Scale Coupling:

Under argon, a solution of 2-7 equivalents, preferably 3 equivalents, ofTCEP in PBS buffer (c ˜0.2-0.8 mg/ml, preferably 0.5 mg/ml) was added to20-200 mg of the antibody in question in PBS buffer (c ˜5-15 mg/ml). Themixture was stirred at RT for 30 min, and then 2-20, preferably 5-10,equivalents of a maleimide precursor compound dissolved in DMSO wereadded. To achieve higher DARs, it is also possible to use 15-20equivalents. After stirring at RT for a further 1.5 h-2 h, the mixturewas diluted with PBS buffer which had been adjusted to pH 8 beforehand.This solution was then applied to PD 10 columns (Sephadex® G-25, GEHealthcare) which had been equilibrated with PBS buffer pH 8 and waseluted with PBS buffer pH 8. The eluate was diluted with PBS buffer pH 8to a concentration of 2-7 mg/ml. This solution was stirred at RT underargon overnight. If required, the solution was then rebuffered to pH7.2. The ADC solution was concentrated by ultracentrifugation, redilutedwith PBS buffer (pH 7.2) and then optionally concentrated again to aconcentration of about 10 mg/ml.

In the structural formulae shown, AK₁ can have the meaning

Examples a: TPP-981 cetuximab (partially reduced)—S§¹

Examples c: TPP-6013 (anti-CD123 AK) (partially reduced)—S§¹

-   -   TPP-8987 (anti-CD123 AK) (partially reduced)—S§¹    -   TPP-8988 (anti-CD123 AK) (partially reduced)—S§¹    -   TPP-9476 (anti-CD123 AK) (partially reduced)—S§¹

Examples e: TPP-1015 (anti-Her2 AK) (partially reduced)—S§¹

Examples h: TPP-8382 (anti-B7H3 AK) (partially reduced)—S§¹

Examples k: TPP-7006 (anti-TWEAKR) (partially reduced)—S§¹

-   -   TPP-7007 (anti-TWEAKR) (partially reduced)—S§¹

Examples x: TPP-9574 (anti-CXCR5 AK) (partially reduced)—S§¹

-   -   TPP-9580 (anti-CXCR5 AK) (partially reduced)—S§¹

wherein

-   -   §¹ represents the linkage to the succinimide group or to any        isomeric hydrolysed open-chain succinamides or the alkylene        radical resulting therefrom,

and

-   -   S represents the sulfur atom of a cysteine residue of the        partially reduced antibody.

B-5. General Process for Coupling to Lysine Side Chains

The following antibodies were used for the coupling reactions:

Examples a: TPP-981 cetuximab (anti-EGFR AK)

Examples c: TPP-6013 (anti-CD123 AK)

-   -   TPP-8987 (anti-CD123 AK)    -   TPP-8988 (anti-CD123 AK)    -   TPP-9476 (anti-CD123 AK)

Examples e: TPP-1015 (anti-Her2 AK)

Examples k: TPP-7006 (anti-TWEAKR AK)

-   -   TPP-7007 (anti-TWEAKR AK)

Examples x: TPP-9574 (anti-CXCR5 AK)

-   -   TPP-9580 (anti-CXCR5 AK)

The coupling reactions were usually carried out under argon.

From 2 to 8 equivalents of the precursor compound to be coupled wereadded as a solution in DMSO to a solution of the antibody in question inPBS buffer in a concentration range between 1 mg/ml and 20 mg/ml,preferably about 10 mg/ml, depending on the intended loading. Afterstirring at RT for 30 min to 6 h, the same amount of precursor compoundin DMSO was added again. Here, the amount of DMSO should not exceed 10%of the total volume. After stirring at RT for a further 30 min to 6 h,the mixture was applied to PD 10 columns (Sephadex® G-25, GE Healthcare)equilibrated with PBS and eluted with PBS buffer. Purification on thePD10 column thus in each case afforded solutions of the respective ADCsin PBS buffer. The sample was then concentrated by ultracentrifugationand optionally rediluted with PBS buffer. If required, for betterremoval of low-molecular weight components, concentration byultrafiltration was repeated after redilution with PBS buffer. Forbiological tests, if required, the concentrations of the final ADCsamples were optionally adjusted to the range of 0.5-15 mg/ml byredilution.

The respective protein concentrations, stated in the working examples,of the ADC solutions were determined. Furthermore, antibody loading(drug/mAb ratio) was determined using the methods described under B-6.

In the structural formulae shown, AK₂ has the meaning

Examples a: TPP-981 cetuximab (anti-EGFR AK)—NH§²

Examples c: TPP-6013 (anti-CD123 AK)—NH§²

-   -   TPP-8987 (anti-CD123 AK)—NH§²    -   TPP-8988 (anti-CD123 AK)—NH§²    -   TPP-9476 (anti-CD123 AK)—NH§²

Examples e: TPP-1015 (anti-Her2 AK)—NH§²

Examples k: TPP-7006 (anti-TWEAKR AK)—NH§²

-   -   TPP-7007 (anti-TWEAKR AK)—NH§²

Examples x: TPP-9574 (anti-CXCR5 AK)—NH§²

-   -   TPP-9580 (anti-CXCR5 AK)—NH§²

wherein

-   -   §² represents the linkage to the carbonyl group

and

-   -   NH represents the side-chain amino group of a lysine residue of        the antibody.

Further Purification and Characterization of the Conjugates According tothe Invention

After the reaction, in some instances the reaction mixture wasconcentrated, for example by ultrafiltration, and then desalted andpurified by chromatography, for example using a Sephadex® G-25 column.Elution was carried out, for example, with phosphate-buffered saline(PBS). The solution was then sterile filtered and frozen. Alternatively,the conjugate can be lyophilized.

B-6. Determination of the Antibody, the Toxophore Loading and theProportion of Open Cysteine Adducts

For protein identification in addition to molecular weight determinationafter deglycosylation and/or denaturing, a tryptic digestion was carriedout which, after denaturing, reduction and derivatization, confirms theidentity of the protein via the tryptic peptides found.

toxophore loading (in the tables referred to as DAR, drug-to-antibodyratio) of the PBS buffer solutions obtained of the conjugates describedin the working examples was determined as follows:

Determination of toxophore loading of lysine-linked ADCs was carried outby mass spectrometry determination of the molecular weights of theindividual conjugate species. Here, the antibody conjugates were firstdeglycosylated with PNGaseF, and the sample was acidified and, afterHPLC separation/desalting, analysed by mass spectrometry usingESI-MicroTof_(Q) (Bruker Daltonik). All spectra over the signal in theTIC (Total Ion Chromatogram) were added and the molecular weight of thedifferent conjugate species was calculated based on MaxEntdeconvolution. The DAR (=drug/antibody ratio) was then calculated aftersignal integration of the different species. For this purpose, the sumtotal of the integration results for all species weighted by thetoxophore count was divided by the sum total of the simply weightedintegration results for all species.

The toxophore loading of cysteine-linked conjugates was determined byreversed-phase chromatography of the reduced and denatured ADCs.Guanidinium hydrochloride (GuHCl) (28.6 mg) and a solution ofDL-dithiothreitol (DTT) (500 mM, 3 μl) were added to the ADC solution (1mg/ml, 50 μl). The mixture was incubated at 55° C. for one hour andanalysed by HPLC.

HPLC analysis was carried out on an Agilent 1260 HPLC system withdetection at 220 nm. A Polymer Laboratories PLRP-S polymericreversed-phase column (catalogue number PL1912-3802) (2.1 ×150 mm, 8 μmparticle size, 1000 Å) was used at a flow rate of 1 ml/min with thefollowing gradient: 0 min, 25% B; 3 min, 25% B; 28 min, 50% B. Eluent Aconsisted of 0.05% trifluoroacetic acid (TFA) in water, eluent B of0.05% trifluoroacetic acid in acetonitrile.

The detected peaks were assigned by retention time comparison with thelight chain (L0) and the heavy chain (H0) of the non-conjugatedantibody. Peaks detected exclusively in the conjugated sample wereassigned to the light chain with one toxophore (L1) and the heavy chainswith one, two and three toxophores (H1, H2, H3).

Average loading of the antibody with toxophores (referred to as DAR,drug-to-antibody ratio) was calculated from the peak areas determined byintegration as double the sum of HC load and LC load, where LC load iscalculated from the sum of the toxophore number-average weighedintegration results of all LC peaks divided by the sum of the singlyweighed integration results of all LC peaks, and where the HC load iscalculated from the sum of the toxophore number-average weighedintegration results of all HC peaks divided by the sum of the singlyweighed integration results of all HC peaks. In individual cases, it wasbe possible that, owing to co-elution of some peaks, it was not possibleto determine toxophore loading accurately.

In the cases where light and heavy chains could not be separatedsufficiently by HPLC, determination of toxophore loading ofcysteine-linked conjugates was carried out by mass spectrometrydetermination of the molecular weights of the individual conjugatespecies at light and heavy chain.

For this purpose, guanidinium hydrochloride (GuHCI) (28.6 mg) and asolution of DL-dithiothreitol (DTT) (500 mM, 3 μl) were added to the ADCsolution (1 mg/ml, 50 μl). The mixture was incubated for one hour at 55°C. and analysed by mass spectrometry after online desalting usingESI-MicroTofQ (Bruker Daltonik).

For the DAR (drug-to-antibody ratio) determination, all spectra wereadded over the signal in the TIC (Total Ion Chromatogram), and themolecular weight of the different conjugate species at light and heavychain was calculated based on MaxEnt deconvolution. The average loadingof the antibody with toxophores was determined from the peak areasdetermined by integration as twice the sum total of the HC loading andthe LC loading. In this context, the LC loading is calculated from thesum total of the integration results for all LC peaks weighted by thetoxophore count, divided by the sum total of the simply weightedintegration results for all LC peaks, and the HC loading from the sumtotal of the integration results for all HC peaks weighted by thetoxophore count, divided by the sum total of the simply weightedintegration results for all HC peaks.

In the case of the open constructs, to determine the proportion of theopen cysteine adduct, the molecular weight area ratio of closed to opencysteine adduct (molecular weight delta 18 daltons) of all singlyconjugated light and heavy chain variants was determined. The mean ofall variants yielded the proportion of the open cysteine adduct.

B-7. Verification of the Antigen Binding of the ADCs

The capability of the binder of binding to the target molecule waschecked after coupling had taken place. The person skilled in the art isfamiliar with various methods which can be used for this purpose; forexample, the affinity of the conjugate can be checked using ELISAtechnology or surface plasmon resonance analysis (BIAcore™ measurement).The conjugate concentration can be measured by the person skilled in theart using customary methods, for example for antibody conjugates byprotein determination. (see also Doronina et al.; Nature Biotechnol.2003; 21:778-784 and Polson et al., Blood 2007; 1102:616-623).

Working Examples of Metabolites

Example M1

N-{(2S)-2-Amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butanoyl}-beta-alanyl-D-glutamicacid

Intermediate C110D was converted into the title compound by a 1-hourhydrogenation over 10% palladium on activated carbon in ethanol underhydrogen standard pressure at RT.

LC-MS (Method 1): R_(t)=1.78 min; MS (ESIpos): m/z=714 [M+H]⁺.

The ADCs shown below in an exemplary manner are capable of releasing thepreferred metabolite Ml, which has preferred pharmacological properties.

Working Examples ADCs

The ADCs shown in the structural formulae of the working examples, whichwere coupled to the cysteine side chains of the antibodies via maleimideradicals, are, depending on the linker and the coupling procedure,mainly present in the ring-opened or ring-closed forms shown in eachcase. However, the preparation may comprise a small proportion of therespective other form.

The coupling reactions were carried out under argon. All the largerbatches for in vivo tests were sterile-filtered at the end of thepreparation.

Examples 1

Exemplary Procedure A:

Under argon, a solution of 0.029 mg of TCEP in 0.05 ml of PBS buffer wasadded to 5 mg of the antibody in question in 0.5 ml of PBS (c=10 mg/ml).The mixture was stirred at RT for 30 min, and then 0.26 mg (0.00023mmol) of Intermediate Q1 dissolved in 50 μl of DMSO was added. Afterstirring at RT for a further 90 min, the mixture was diluted to a volumeof 2.5 ml with PBS buffer which had been adjusted to pH 8 beforehand andthen passed through a PD 10 column (Sephadex® G-25, GE Healthcare)equilibrated with PBS buffer pH 8, and eluted with PBS buffer pH 8. Theeluate was stirred at RT under argon overnight. This was followed byconcentration by ultracentrifugation and redilution with PBS buffer (pH7.2).

Exemplary Procedure B:

Under argon, a solution of 0.172 mg of TCEP in 0.3 ml of PBS buffer wasadded to 30 mg of the antibody in question in 3 ml of PBS (c=10 mg/ml).The mixture was stirred at RT for 30 min, and then 1.57 mg (0.0014 mmol)of Intermediate Q1 dissolved in 300 μl of DMSO was added. After stirringat RT for a further 90 min, the mixture was diluted to a volume of 5 mlwith PBS buffer which had been adjusted to pH 8 beforehand and thenpassed through a PD 10 column (Sephadex® G-25, GE Healthcare)equilibrated with PBS buffer pH 8, and eluted with PBS buffer pH 8. Theeluate was diluted with PBS buffer pH 8 to a volume of 7.5 ml andstirred at RT under argon overnight. This solution was then applied to aPD 10 column (Sephadex® G-25, GE Healthcare) which had been equilibratedwith PBS buffer pH 7.2 and was eluted with PBS buffer pH 7.2. The eluatewas then concentrated by ultracentrifugation, rediluted with PBS buffer(pH 7.2) and reconcentrated and sterile-filtered again.

The following ADCs were prepared analogously to these procedures andcharacterized as indicated in the table:

Antibody Example Target TPP- Procedure C [mg/ml] DAR 1a-981 EGFR 981 A1.85 2.5 1c-6013 CD123 6013 A 2.0 2.4 1c-9476 CD123 9476 A 1.96 3.11e-1015 HER2 1015 A 1.75 3.3 1h-8382 B7H3 8382 B 11.01 3.5 1k-7006TWEAKR 7006 A 1.8 2.9 1k-7007 TWEAKR 7007 B 7.84 3.3 1x-9574 CXCR5 9574A 1.26 2.9

Examples 2

Exemplary Procedure A:

Under argon, 5 eq (0.2 mg) of Intermediate Q2 dissolved in 50 μl of DMSOwere added to 5 mg of the antibody in question in 0.5 ml of PBS (c=10mg/ml). After stirring at RT for 1 h, the same amount again was addedand the mixture was stirred at RT for a further hour. The reactionmixture was subsequently diluted to 2.5 ml with PBS buffer (pH 7.2),purified on a Sephadex column, then concentrated by ultracentrifugationand rediluted with PBS (pH 7.2).

Exemplary Procedure B:

Under argon, 4 eq (1 mg) of Intermediate Q2 dissolved in 50 μl of DMSOwere added to 30 mg of the antibody in question in 3 ml of PBS buffer(pH 7.2) (c=10 mg/ml). After stirring at RT for 1 h, the same amountagain was added and the mixture was stirred at RT for a further hour.Then the mixture was diluted to 5 ml with PBS buffer (pH 7.2), purifiedon a Sephadex column, then concentrated by ultracentrifugation,rediluted with PBS (pH 7.2) and reconcentrated and sterile-filteredagain.

Exemplary Procedure C:

Under argon, 2.5 eq (1 mg) of Intermediate Q2 dissolved in 250 μl ofDMSO were added to 50 mg of the antibody in question in 5 ml of PBSbuffer (pH 7.2) (c=10 mg/ml). After stirring at RT for 1 h, the sameamount again was added and the mixture was stirred at RT for a furtherhour. Then the mixture was diluted to 7.5 ml with PBS buffer (pH 7.2),purified on a Sephadex column, then concentrated by ultracentrifugation,rediluted with PBS (pH 7.2) and reconcentrated and sterile-filteredagain.

Exemplary Procedure D:

Under argon, 4.5 eq (36 mg) of Intermediate Q2 dissolved in 7.5 ml ofDMSO were added to 1000 mg of the antibody in question in 150 ml of PBSbuffer (pH7.2) (c=6.7 mg/ml). After stirring at RT for 1 h, the sameamount again was added and the mixture was stirred at RT for a furtherhour. The batch was then purified by cross-flow filtration, concentratedand sterile-filtered.

Antibody Example Target TPP- Procedure C [mg/ml] DAR 2a-981 EGFR 981 A2.23 4.5 2c-6013 CD123 6013 A 2.32 4.9 2c-8987 CD123 8987 B 8.86 5.82c-8988 CD123 8988 B 9.81 3.6 2c-9476B CD123 9476 B 10.27 4.2 2c-94760CD123 9476 C 9.22 3.4 2c-9476D CD123 9476 D 15.83 6.3 2e-1015 HER2 1015A 2.05 5.4 2k-7006 TWEAKR 7006 A 2.09 5.9 2k-7007 TWEAKR 7007 B 9.32 3.42x-9574 CXCR5 9574 B 9.5 4.8 2x-9580 CXCR5 9580 B 10.12 4.8

Examples 3

Exemplary Procedure A:

Under argon, 5 eq (0.2 mg) of Intermediate Q3 dissolved in 50 μl of DMSOwere added to 5 mg of the antibody in question in 0.5 ml of PBS (c=10mg/ml). After stirring at RT for 1 h, the same amount again was addedand the mixture was stirred at RT for a further hour. The reactionmixture was subsequently diluted to 2.5 ml with PBS buffer (pH 7.2),purified on a Sephadex column, then concentrated by ultracentrifugationand rediluted with PBS (pH 7.2).

Exemplary Procedure B:

Under argon, 4 eq (1 mg) of Intermediate Q3 dissolved in 50 μl of DMSOwere added to 30 mg of the antibody in question in 3 ml of PBS buffer(pH 7.2) (c=10 mg/ml). After stirring at RT for 1 h, the same amountagain was added and the mixture was stirred at RT for a further hour.Then the mixture was diluted to 2.5 ml with PBS buffer (pH 7.2),purified on a Sephadex column, then concentrated by ultracentrifugation,rediluted with PBS (pH 7.2) and reconcentrated and sterile-filteredagain.

Antibody Example Target TPP- Procedure C [mg/ml] DAR 3a-981 EGFR 981 A1.99 5.0 3c-9476 CD123 9476 A 2.09 5.8 3e-1015 HER2 1015 A 2.06 6.33k-7007 TWEAKR 7007 A 2.11 5.3 3x-9574 CXCR5 9574 A 2.02 4.5

Examples 4

Exemplary Procedure A:

Under argon, a solution of 0.029 mg of TCEP in 0.05 ml of PBS buffer wasadded to 5 mg of the antibody in question in 0.4 ml of PBS buffer (pH7.2) (c=12.5 mg/ml). The mixture was stirred at RT for 30 min, and then0.275 mg (0.00023 mmol) of Intermediate Q4 dissolved in 50 μl of DMSOwas added. After a further 90 min of stirring at RT, the reaction wasdiluted to a total volume of 2.5 ml with PBS buffer. This solution wasthen applied to a PD 10 column (Sephadex® G-25, GE Healthcare) which hadbeen equilibrated with PBS buffer (pH 7.2) and was eluted with PBSbuffer (pH 7.2). This was followed by concentration byultracentrifugation and redilution with PBS buffer (pH 7.2).

Antibody Example Target TPP- Procedure C [mg/ml] DAR 4c-9476 CD123 9476A 2.06 3.5 4k-7007 TWEAKR 7007 A 1.87 4.0 4x-9574 CXCR5 9574 A 1.93 3.6

Examples 5

Exemplary Procedure A:

Under argon, 5 eq (0.2 mg) of Intermediate Q5 dissolved in 50 μl of DMSOwere added to 5 mg of the antibody in question in 0.5 ml of PBS (c=10mg/ml). After stirring at RT for 1 h, the same amount again was addedand the mixture was stirred at RT for a further hour. The reactionmixture was subsequently diluted to 2.5 ml with PBS buffer (pH 7.2),purified on a Sephadex column, then concentrated by ultracentrifugationand rediluted with PBS (pH 7.2).

Antibody Example Target TPP- Procedure C [mg/ml] DAR 5c-9476 CD123 9476A 2.37 5.3 5e-1015 HER2 1015 A 2.33 5.5 5k-7007 TWEAKR 7007 A 2.18 5.65x-9574 CXCR5 9574 A 1.88 6.8

Examples 6

Exemplary Procedure A:

Under argon, 5 eq (0.18 mg) of Intermediate Q6 dissolved in 50 μl ofDMSO were added to 5 mg of the antibody in question in 0.4 ml of PBS(c=12.5 mg/ml). After stirring at RT for 1 h, the same amount again wasadded and the mixture was stirred at RT for a further hour. The reactionmixture was subsequently diluted to 2.5 ml with PBS buffer (pH 7.2),purified on a Sephadex column, then concentrated by ultracentrifugationand rediluted with PBS (pH 7.2).

Antibody Example Target TPP- Procedure C [mg/ml] DAR 6a-981 EGFR 981 A2.39 4.9 6c-9476 CD123 9476 A 1.8 5.3 6e-1015 HER2 1015 A 2.23 6.26k-7007 TWEAKR 7007 A 2.57 5.6

Examples 7

Exemplary Procedure A:

Under argon, a solution of 0.029 mg of TCEP in 0.05 ml of PBS buffer wasadded to 5 mg of the antibody in question in 0.4 ml of PBS (c=12.5mg/ml). The mixture was stirred at RT for 30 min, and then 0.24 mg(0.00023 mmol) of Intermediate Q7 dissolved in 50 μl of DMSO was added.After stirring at RT for a further 90 min, the mixture was diluted to avolume of 2.5 ml with PBS buffer which had been adjusted to pH 8beforehand and then passed through a PD 10 column (Sephadex® G-25, GEHealthcare) equilibrated with PBS buffer pH 8, and eluted with PBSbuffer pH 8. The eluate was stirred at RT under argon overnight. Thiswas followed by concentration by ultracentrifugation and redilution withPBS buffer (pH 7.2).

Antibody C Example Target TPP- Procedure [mg/ml] DAR 7a-981 EGFR 981 A2.03 3.4 7c-9476 CD123 9476 A 1.53 4.0 7e-1015 HER2 1015 A 1.88 3.87k-7007 TWEAKR 7007 A 1.99 3.6

Examples 8

Exemplary Procedure A:

Under argon, 5 eq (0.2 mg) of Intermediate Q8 dissolved in 50 μl of DMSOwere added to 5 mg of the antibody in question in 0.5 ml of PBS (c=10mg/ml). After stirring at RT for 1 h, the same amount again was addedand the mixture was stirred at RT for a further hour. The reactionmixture was subsequently diluted to 2.5 ml with PBS buffer (pH 7.2),purified on a Sephadex column, then concentrated by ultracentrifugationand rediluted with PBS (pH 7.2).

Antibody C Example Target TPP- Procedure [mg/ml] DAR 8a-981 EGFR 981 A2.32 6.5 8c-9476 CD123 9476 A 2.37 6.9 8e-1015 HER2 1015 A 1.46 6.68k-7007 TWEAKR 7007 A 2.43 6.7

Examples 9

Exemplary Procedure A:

Under argon, 5 eq (0.2 mg) of Intermediate Q9 dissolved in 50 μl of DMSOwere added to 5 mg of the antibody in question in 0.5 ml of PBS (c=10mg/ml). After stirring at RT for 1 h, the same amount again was addedand the mixture was stirred at RT for a further hour. The reactionmixture was subsequently diluted to 2.5 ml with PBS buffer (pH 7.2),purified on a Sephadex column, then concentrated by ultracentrifugationand rediluted with PBS (pH 7.2).

Antibody C Example Target TPP- Procedure [mg/ml] DAR 9a-981 EGFR 981 A2.34 4.4 9c-9476 CD123 9476 A 2.65 4.2 9e-1015 HER2 1015 A 2.22 4.89k-7007 TWEAKR 7007 A 2.14 3.8

Examples 10

Exemplary Procedure A:

Under argon, a solution of 0.029 mg of TCEP in 0.05 ml of PBS buffer wasadded to 5 mg of the antibody in question in 0.5 ml of PBS buffer (pH7.2) (c=10 mg/ml). The mixture was stirred at RT for 30 min, and then0.295 mg (0.00023 mmol) of Intermediate Q10 dissolved in 50 μl of DMSOwas added. After a further 20 h of stirring at RT, the reaction wasdiluted to a total volume of 2.5 ml with PBS buffer. This solution wasthen applied to a PD 10 column (Sephadex® G-25, GE Healthcare) which hadbeen equilibrated with PBS buffer (pH 7.2) and was eluted with PBSbuffer (pH 7.2). This was followed by concentration byultracentrifugation and redilution with PBS buffer (pH 7.2).

Exemplary Procedure C for Obtaining a Higher DAR:

Under argon, a solution of 0.057 mg of TCEP in 0.05 ml of PBS buffer wasadded to 5 mg of the antibody in question in 0.5 ml of PBS buffer (pH7.2) (c=10 mg/ml). The mixture was stirred at RT for 30 min, and then0.59 mg (0.00053 mmol) of Intermediate Q10 dissolved in 50 μl of DMSOwas added. After a further 20 h of stirring at RT, the reaction wasdiluted to a total volume of 2.5 ml with PBS buffer. This solution wasthen applied to a PD 10 column (Sephadex® G-25, GE Healthcare) which hadbeen equilibrated with PBS buffer (pH 7.2) and was eluted with PBSbuffer (pH 7.2). This was followed by concentration byultracentrifugation and redilution with PBS buffer (pH 7.2).

Antibody C Example Target TPP- Procedure [mg/ml] DAR 10a-981 EGFR 981 A1.9 3.2 10c-9476 CD123 9476 A 1.83 2.7 10c-9476 CD123 9476 C 1.97 4.8 hD10e-1015 HER2 1015 A 1.83 3.4 10k-7007 TWEAKR 7007 A 1.9 4.7 10x-9574CXCR5 9574 A 1.41 3.8 10x-9574 CXCR5 9574 C 0.97 6.3 hD

Examples 11

Exemplary Procedure A:

Under argon, a solution of 0.029 mg of TCEP in 0.05 ml of PBS buffer wasadded to 5 mg of the antibody in question in 0.4 ml of PBS buffer (pH7.2) (c=12.5 mg/ml). The mixture was stirred at RT for 30 min, and then0.32 mg (0.00023 mmol) of Intermediate Q11 dissolved in 50 μl of DMSOwas added. After a further 20 h of stirring at RT, the reaction wasdiluted to a total volume of 2.5 ml with PBS buffer. This solution wasthen applied to a PD 10 column (Sephadex® G-25, GE Healthcare) which hadbeen equilibrated with PBS buffer (pH 7.2) and was eluted with PBSbuffer (pH 7.2). This was followed by concentration byultracentrifugation and redilution with PBS buffer (pH 7.2).

Antibody C Example Target TPP- Procedure [mg/ml] DAR 11a-981 EGFR 981 A1.88 1.9 11c-9476 CD123 9476 A 1.89 1.6 11e-1015 HER2 1015 A 1.69 2.311k-7007 TWEAKR 7007 A 1.17 1.9

For Comparative Purposes, the Following ADCs were Prepared:

Reference Example R1

Such ADCs were disclosed in WO2015/096982 and in WO2016/096610 withvarious antibodies including, for example, cetuximab and trastuzumab.For comparative purposes, the precursor Intermediate F194 disclosedtherein was furthermore also reacted with TPP-6013 (anti-CD123 AK). Thefollowing ADCs were used for comparative purposes:

Antibody C Example Target TPP- [mg/ml] DAR R1a EGFR 981 1.67 1.9 R1cCD123 6013 0.42 2.9 R1e HER2 1015 1.39 2.4 R1x CXCR5 9574 1.28 2.2

Reference Example R2

Such ADCs were disclosed in WO2016/096610 with an aglycosylatedanti-TWEAKR antibody. For comparative purposes, the precursorIntermediate F291 disclosed therein was furthermore also reacted withTPP-9574 (anti-CXCR5 AK), TPP-981 (anti-EGFR) and TPP-1015 (anti-HER2AK). The following ADCs were used for comparative purposes:

Antibody C Example Target TPP- [mg/ml] DAR R2a EGFR 981 1.46 3.4 R2eHER2 1015 1.42 3.5 R2x CXCR5 9574 1.41 3.6

For the Reference Examples R1, WO2015/096982 describes the metaboliteExample 98 derived therefrom. For the Reference Examples R2,WO2016/096610 describes the identical metabolite Example M9, which islisted here as Reference Example R3M.

Reference Example R3MN-(3-Aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide

The preparation was described in WO2015/096982 as Example 98.

The biological data for these reference compounds disclosed in saidapplications or obtained for the novel reference compounds are describedin Chapter C.

C: Assessment of Biological Efficacy

The biological activity of the compounds according to the invention canbe shown in the assays described below:

a. C-1a Determination of the Cytotoxic Effect of the ADCs

The analysis of the cytotoxic effect of the ADCs was carried out withvarious cell lines:

NCI-H292: human mucoepidermoid lung carcinoma cells, ATCC-CRL-1848,standard medium: RPMI 1640 (Biochrom; #FG1215, stab. glutamine)+10% FCS(Sigma; #F2442), TWEAKR-positive; EGFR-positive.

BxPC3: human pancreas carcinoma cells, ATCC-CRL-1687, standard medium:RPMI 1640 (Biochrom; #FG1215, stab. glutamine)+10% FCS (Sigma; #F2442),TWEAKR-positive.

LoVo: human colorectal cancer cells, ATCC No. CCL-229, cultivation forMTT assay: standard medium: Kaighn's+L-glutamine (Invitrogen 21127)+10%heat inactivated FCS (from Gibco, No. 10500-064). Cultivation for CTGassay: RPMI 1640 (Biochrom; #FG1215, stab. glutamine)+10% FCS (Sigma#F2442). TWEAKR-positive.

KPL4: human breast cancer cell line, Bayer Pharma AG (identity checkedand confirmed on 19.7.2012 at DSMZ), standard medium: RPMI 1640 (fromGibco; #21875-059, stab. L-glutamine)+10% heat inactivated FCS (Gibco,No. 10500-064); HER2-positive.

SK-HEP-1: human liver cell cancer line, ATCC No. HTB-52, standardmedium: MEM with Earle's salts+Glutamax I (Invitrogen 41090)+10% heatinactivated FCS (from Gibco, No. 10500-064); EGFR-positive,TWEAKR-positive

MOLM-13: human acute monocytic leukaemia cells (AML-M5a), DSMZ, No. ACC554, standard medium: RPMI 1640 (from Gibco; #21875-059, stab.L-glutamine)+20% heat inactivated FCS (Gibco, No. 10500-064);CD123-positive.

MV-4-11: human biphenotypic B myelomonocytic leukaemia cells obtainedfrom peripheral blood, ATCC-CRL-9591, standard medium: IMDM (ATCC:30-2005), +10% heat inactivated FCS (Gibco, No. 10500-064);CD123-positive.

NB4: human acute promyelocytic leukaemia cells obtained from bonemarrow, DSMZ, No. ACC 207, standard medium: RPMI 1640+GlutaMAX I(Invitrogen 61870)+10% heat inactivated FCS (Gibco, No. 10500-064)+2.5 gof glucose (20% glucose solution, Gibco, No.19002)+10 mM Hepes(Invitrogen 15630)+1 mM sodium pyruvate (Invitrogen 11360);CD123-negative

Rec-1: human mantle cell lymphoma cells (B cell non-Hodgkin's lymphoma)ATCC CRL-3004, standard medium: RPMI 1640+GlutaMAX I (Invitrogen61870)+10% heat inactivated FCS (Gibco, No. 10500-064)+10 mM)CXCR5-positive

U251: human glioblastoma cells, standard medium: RPMI 1640 (Biochrom;#FG1215, stab. glutamine)+10% FCS (Biochrom; #S0415), B7H3-positive.

HBL-1: human B cell lymphoma cells (diffuse large B-cell lymphoma) ATTCRL-RRID (Resource Identification Initiative): CVCL_4213, firstdescribed in Abe et al. Cancer 61:483-490(1988), obtained from Prof.Lenz, Universität Münster; standard medium: RPMI 1640 (Biochrom;#FG1215, stab. glutamine)+10% FCS (Biochrom; #S0415), cultivationanalogous to Rec-1 cells; CXCR5 positive

The cells were cultivated by the standard method as stated by theAmerican Tissue Culture Collection (ATCC) or the Leibniz-InstitutDSMZ-Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ)for the cell lines in question.

CTG Assay

The cells were cultivated by the standard method, with the growth mediaspecified under C-1. The test was carried out by detaching the cellswith a solution of trypsin (0.05%) and EDTA (0.02%) in PBS (Biochrom AG#L2143), pelleting, resuspending in culture medium, counting and sowinginto a 96-well culture plate with white bottom (Costar #3610) (at 75μl/well, the following cell numbers per well are: NCI-H292: 2500cells/well, BxPC3 2500 cells/well, LoVo 3000 cells/well) and incubatingin an incubator at 37° C. and 5% carbon dioxide. The suspension cellswere counted and sown in a 96-well culture plate with white bottom(Costar #3610) (at 75 μl/well, the following cell numbers per well:Rec-1: 3000 cells/well, HBL-1: 6000 cells/well). After 24 h, theantibody drug conjugates were added in 25 μl of culture medium(concentrated four-fold) to the cells to give final antibody drugconjugate concentrations of 3×10⁻⁷M to 3×10⁻¹¹M on the cells(triplicates). The cells were then incubated in an incubator at 37° C.and 5% carbon dioxide. On a parallel plate, the cell activity at thestart of the drug treatment (day 0) was determined using the Cell TiterGlow (CTG) luminescent cell viability assay (Promega #G7573 and #G7571).To this end, per cell batch 100 μl of the substrate were added, theplates were then covered with aluminium foil, shaken on the plate shakerat 180 rpm for 2 minutes, allowed to stand on the laboratory bench for 8minutes and then measured using a luminometer (Victor X2, Perkin Elmer).The substrate detects the ATP content in the living cells generating aluminescence signal whose intensity is directly proportional to theviability of the cells. After 72 h of incubation with the antibody drugconjugates, the viability of these cells was then also determined usingthe Cell Titer Glow luminescent cell viability assay as described above.From the data measured, the IC₅₀ of the growth inhibition was calculatedin comparison to day 0 using the DRC (Dose Response Curve) analysisspreadsheets and a 4-parameter fit. The DRC analysis spreadsheet is abiobook spreadsheet developed by Bayer Pharma AG and Bayer BusinessServices on the IDBS E-WorkBook Suite platform (IDBS: ID BusinessSolutions Ltd., Guildford, UK).

MTT Assay

The cells were cultivated by the standard method, with the growth mediaspecified under C-1. The test was carried out by detaching the cellswith a solution of Accutase in PBS (from Biochrom AG #L2143),pelletizing, resuspending in culture medium, counting and sowing into a96-well culture plate with white bottom (from Costar #3610) (NCI H292:2500 cells/well; SK-HEP-1: 1000 cells/well; KPL-4: 1200 cells/well; intotal volume 100 μl). The cells were then incubated in an incubator at37° C. and 5% carbon dioxide. After 48 h, the medium was replaced. Theantibody drug conjugates in 10 μl of culture medium in concentrationsfrom 10⁻⁵M to 10⁻¹³M were then pipetted to the cells (in triplicate),and the assay was then incubated in an incubator at 37° C. and 5% carbondioxide. The suspension cells were counted and sown into a 96-wellculture plate with white bottom (from Costar #3610) (#3610) (MOLM-13:2000 cells/well; NB4: 7000 cells/well; MV-4-11: 5000 cells/well in atotal volume of 100 μl). After 6 hours of incubation at 37° C. and 5%carbon dioxide, the medium was changed and the antibody-drug conjugatesor metabolites were added by pipette in 10 μl of culture medium inconcentrations of 10⁻⁵M to 10⁻¹³M to the cells (triplicates) in 90 μl.The batch was incubated in an incubator at 37° C. and 5% carbon dioxide.After 96 h, the cell proliferation was detected using the MTT assay(ATCC, Manassas, Va., USA, catalogue No. 30-1010K). To this end, the MTTreagent was incubated with the cells for 4 h, followed by lysis of thecells overnight by addition of the detergent. The dye formed wasdetected at 570 nm (Infinite M1000 pro, Tecan). The measured data wereused to calculate the IC₅₀ of the growth inhibition using the DRC (doseresponse curve). The proliferation of cells which were not treated withtest substance but were otherwise identically treated was defined as the100% figure.

Tables 1a and 1b below set out the IC₅₀ values for representativeworking examples from these assays:

TABLE 1a MOLM MV-4- NCI- 13 11 H292 LoVo SKHep- BxPC3 KPL-4 IC₅₀ IC₅₀IC₅₀ [M] IC₅₀ 1 IC₅₀ IC₅₀ Exam- [M] [M] MTT/ [M] IC₅₀ [M] [M] [M] pleMTT MTT CTG CTG MTT CTG [MTT] 1a-981 2.65E− 6.70E− 11 08 1c-6013 3.15E−2.29E− 10 08 1c-9476 2.28E− 3.19E− 09 09 1e-1015 1.51E− 09 1k-70061.12E− 5.37E− 3.62E− 1.04E− 10 11 11 10 1k-7007 6.56E− 6.70E− 8.42E−9.61E− 11 11 11 11 2a-981 5.50E− 1.79E− 12 10 2c-6013 1.78E− 4.77E− 1110 2c-8987 4.59E− 1.26E− 10 10 2c-8988 8.91E− 4.39E− 11 09 2c- 9.15E−5.08E− 9476B 10 10 2c- 1.3E−08 2.01E− 9476C 09 2c- 1.10E− 6,80E− 9476D09 11 2e-1015 1.43E− 10 2k-7006 5.86E− 1.50E− 2.04E− 9.92E− 11 11 11 112k-7007 8.82E− 1.50E− 7.54E− 11 11 11 3a-981 7.73E− 1.11E− 13 10 3c-94762.00E− 1.05E− 10 11 3e-1015 3.64E− 11 3k-7007 5.49E− 1.50E− 9.80E− 11 1111 4a-981 8.40E− 1.57E− 10 10 4c-9476 8.40E− 1.57E− 10 10 4k-7007 7.74E−1.50E− 1.18E− 10 11 10 5c-9476 1.08E− 4.40E− 09 11 5e-1015 4.50E− 105k-7007 1.12E− 1.50E− 3.03E− 10 11 10 5x-9574 6a-981 1.68E− 5.00E− 12 076c-9476 4.46E− 1.24E− 10 10 6e-1015 5.54E− 10 6k-7007 6.94E− 4.93E−1.89E− 11 11 10 7a-981 2.34E− 10 7c-9476 1.09E− 6.4E− 09 10 7e-10154.51E− 10 7k-7007 1,03E− 1.43E− 4.39E− 10 09 10 8a-981 9,87E− 3.96E− 1208 8c-9476 3,36E− 3.46E− 10 11 8e-1015 4.30E− 10 8k-7007 1.84E− 3.66E−4.17E− 10 11 10 9a-981 1.00E− 4.29E− 11 08 9c-9476 5.00E− 1.44E− 07 079e-1015 1.39E− 10 9k-7007 1.45E− 3.17E− 2.69E− 10 11 10 10a-981 1.00E−5.00E− 12 07 10c- 1,41E− 8,42E− 9476 09 10 10c- 1.79E− 5.45E− 9476 hD 1011 10e- 3.78E− 1015 11 10k- 9.60E− 1.50E− 1.29E− 7007 11 11 10 11a-9811.98E− 2.59E− 12 08 11c- 9.61E− 6.90E− 9475 08 08 11e- 2.37E− 1015 1011k- 1.74E− 1.62E− 7007 10 10

TABLE 1b U251 HBL1 Rec-1 IC₅₀ IC₅₀ IC₅₀ [M] [M] [M] Example CTG CTG CTG1h-8382 9.98E−10 1x-9574 1.50E−11 7.69E−9  2x-9574  1.5E−11 2.76E−102x-9580  7.7E−11 3x-9574 1.50E−11 4x-9574 1.50E−11 6.17E−11 5x-95741.50E−11 10x-9574 1.50E−11 2.76E−9  10x-9574 hD 1.50E−11 4.64E−11

Table 1c below lists the IC₅₀ values for the reference examples fromthese assays.

TABLE 1c MOLM 13 Rec-1 IC₅₀ NCI-H292 SKHep-1 KPL-4 IC₅₀ [M] [M] IC₅₀ [M]IC₅₀ [M] IC₅₀ [M] Example MTT CTG MTT MTT [MTT] R1a 6.14E−11 1.85E−10R1c 1.24E−07 R1e 1.55E−08 R1x 3.00E−07 R2a 2.10E−10 6.02E−08 R2e4.39E−08 R2x 3.00E−07

The activity data reported relate to the working examples described inthe present experimental section, with the drug/mAB ratios indicated.The values may possibly deviate for different drug/mAB ratios. The IC50values are means of several independent experiments or individualvalues. The action of the antibody drug conjugates was selective for therespective isotype control comprising the respective linker andtoxophore. For the ADCs directed against CD123, the target specificitywas additionally demonstrated by testing with a CD123-negative cell.

In general, the ADCs according to the invention exhibit significantlyimproved cytotoxic potency compared to the corresponding referenceexamples.

C-1b Determination of the Inhibition of the Kinesin Spindle ProteinKSP/EQ5 by Selected Examples

The motor domain of the human kinesin spindle protein KSP/Eg5(tebu-bio/Cytoskeleton Inc, No. 027EG01-XL) was incubated in aconcentration of 10 nM with microtubuli (bovine or porcine,tebu-bio/Cytoskeleton Inc) stabilized with 50 μg/ml taxol (Sigma No.T7191-5MG) for 5 min at RT in 15 mM PIPES, pH 6.8 (5 mM MgCl₂ and 10 mMDTT, Sigma). The freshly prepared mixture was aliquoted into a 384 MTP(from Corning). The inhibitors to be examined at concentrations of1.0×10-6 M to 1.0×10-13 M and ATP (final concentration 500 μM, Sigma)were then added. Incubation was at RT for 2 h. ATPase activity wasdetected by detecting the inorganic phosphate formed using malachitegreen (Biomol). After addition of the reagent, the assay was incubatedat RT for 50 min prior to detection of the absorption at a wavelength of620 nm. The positive controls used were monastrol (Sigma, M8515-1mg) andispinesib (AdooQ Bioscience A10486). The individual data of thedose-activity curve are eight-fold determinations. The IC₅₀ values aremeans of two independent experiments. The 100% control was the samplewhich had not been treated with inhibitors.

Table 2 below lists the IC₅₀ values of representative working examplesfrom the assay described and summarizes the corresponding cytotoxicitydata (MTT assay):

TABLE 2 KSP NCI-H292 SK-Hep-1 KPL4 MOLM-13 assay IC₅₀ [M] IC₅₀ [M] IC₅₀[M] IC₅₀ [M] Examples IC₅₀ [M] MTT MTT MTT MTT M1 1.59E−09 1.74E−071.96E−08 1.73E−07 R3M 1.09E−09 2.70E−10 7.62E−09 2.57E−10 6.69E-11

The activity data reported relate to the working examples described inthe present experimental section.

C-1c Enzymatic Assays

a: Cathepsin B Assay

For every cathepsin B-cleavable prodrug to be examined, a mixture wasmade up in a micro reaction vessel (0.5 ml, from Eppendorf). The enzymeused here was obtained from human liver tissue. 2 μg of cathepsin B(Sigma C8571 25 μg) were initially charged and made up to a total volumeof 200 μl with 50 mM Na phosphate buffer, pH6.0, 2 mM DTT. Then 50 μl ofthe substrate solution to be examined were pipetted in. The mixture wasincubated in a thermoblock (from Thermo Fisher Scientific) at 40° C.under constant agitation at 300 rpm. The enzymatic reaction wascontrolled kinetically. For this purpose, a 10 μl sample was taken atdifferent times. The sample taken was admixed immediately with 20 μl ofice-cold methanol in order to stop the enzymatic reaction and thenfrozen at −20° C. The times selected for sampling were after 10 min, 2h, 4 h and 24 h. The samples were examined by RP-HPLC analysis (reversephase HPLC, Agilent Technologies 1200 Series). The determination of thetoxophore released enabled the determination of the half-life t_(1/2) ofthe enzymatic reaction.

b: Legumain Assay

The legumain assay was conducted with recombinant human enzyme. Therhlegumain enzyme solution (catalogue #2199-CY, R&D Systems) was dilutedin 50 mM Na acetate buffer/100 mM NaCl, pH 4.0 to the desiredconcentration and preincubated at 37° C. for 2 h. rhLegumain was thenadjusted to a final concentration of 1 ng/μl in 50 mM MES buffer, 250 mMNaCl, pH 5.0. For every legumain-cleavable prodrug to be examined, amixture was made up in a micro reaction vessel (0.5 ml, from Eppendorf).For this purpose, the substrate solution was adjusted to the desiredconcentration (double concentration) with 50 mM MES buffer, 250 mM NaCl,pH 5.0. For the kinetic measurement of the enzymatic reaction, 250 μl ofthe legumain solution were first initially charged and the enzymereaction was started by adding 250 μl of the substrate solution (finalconcentration: single concentration; 3 μM). At various points in time,50 μl samples were taken. Immediately, 100 μl of ice-cold methanol wereadded to this sample in order to stop the enzymatic reaction, and thesample was then frozen at −20° C. The times selected for sampling wereafter 0.5 h, 1 h, 3 h and 24 h. The samples were then analysed by meansof RP-HPLC analysis and by LC-MS analysis. The determination of thetoxophore released enabled the determination of the half-life t_(1/2) ofthe enzymatic reaction.

As representative examples to show the legumain-mediated cleavage, thesubstrates produced in the legumain assay were the model compounds A andB.

Reference Example Model Compound A

N-(Pyridin-4-ylacetyl)-L-alanyl-L-alanyl-N¹-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-1-(methylamino)-1-oxobutan-2-yl]-L-aspartamide

First of all, trifluoroacetic acid(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-N-methylbutanamidewas prepared as described in WO 2015096982 A1. Subsequently, thisintermediate was used to prepare the title compound by coupling toIntermediate L103 in DMF in the presence of HATU and ofN,N-diisopropylethylamine.

LC-MS (Method 1): R_(t)=0.86 min; MS (ESIpos): m/z=902 [M+H]⁺.

Reference Example Model Compound B

N-(Pyridin-4-ylacetyl)-L-alanyl-N-methyl-L-alanyl-N¹-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-1-(methylamino)-1-oxobutan-2-yl]-L-aspartamide

First of all, trifluoroacetic acid(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-N-methylbutanamidewas prepared as described in WO 2015096982 A1. Subsequently, thisintermediate was used to prepare the title compound by coupling toIntermediate L118 in DMF in the presence of HATU and ofN,N-diisopropylethylamine.

LC-MS (Method 1): R_(t)=0.83 min; MS (ESIpos): m/z=916 [M+H]⁺.

Model compound A was cleaved under the conditions described above forlegumain to the target compound with a half-life of 0.4 h.

Model compound B was cleaved under the conditions described above forlegumain to the target compound with a half-life of 0.5 h.

C-2 Internalization Assay

Internalization is a key process which enables specific and efficientprovision of the cytotoxic payload in antigen-expressing cancer cellsvia antibody drug conjugates (ADC). This process is monitored viafluorescent labelling of specific antibodies and an isotype controlantibody. First, the fluorescent dye was conjugated to lysines of theantibody. Conjugation was carried out using a two-fold to 10-fold molarexcess of CypHer 5E mono NHS ester (Batch 357392, GE Healthcare) at pH8.3. After the coupling, the reaction mixture was purified by gelchromatography (Zeba Spin Desalting Columns, 40K, Thermo Scientific, No.87768; elution buffer: DULBECCO'S PBS, Sigma-Aldrich, No. D8537), toeliminate excess dye and to adjust the pH. The protein solution wasconcentrated using VIVASPIN 500 columns (Sartorius stedim biotec). Thedye load of the antibody was determined by means of spectrophotometryanalysis (from NanoDrop) and subsequent calculation (D/P=A_(dye)ε_(protein):(A₂₈₀-0.16 A_(dye))ε_(dye)).

The dye load of the antibodies examined here and the isotype controlwere of a comparable order of magnitude. In cell binding assays, it wasconfirmed that the coupling did not lead to any change in the affinityof the antibodies.

The labelled antibodies were used for the internalization assay. Priorto the start of the treatment, cells (2×10⁴/well) were sown in 100 μlmedium in a 96-well MTP (fat, black, clear bottom No 4308776, fromApplied Biosystems). After 18 h of incubation at 37° C./5% CO₂, themedium was replaced and labelled antibodies were added in differentconcentrations (10, 5, 2.5, 1, 0.1 μg/ml). The same treatment protocolwas applied to the labelled isotype control (negative control). Thechosen incubation times were 0 h, 0.25 h, 0.5 h, 1 h, 1.5 h, 2 h, 3 h, 6h and 24 h. The fluorescence measurement was carried out using theInCellAnalyzer 1000 (from GE Healthcare). This was followed by kineticevaluation via measurement of the parameters granule counts/cell andtotal granule intensity/cell.

Following binding to the receptor, antibodies were examined for theirinternalization capacity. For this purpose, cells with differentreceptor expression levels were chosen. A target-mediated specificinternalization was observed with the antibodies, whereas the isotypecontrol showed no internalization.

C-2b Internalization Assay with Suspended Cells

Coupling of the fluorescent dye was carried out as described under C2.The antigen to be examined is expressed by haematopoietic suspensioncells; consequently, the internalization was examined in an FACS-basedinternalization assay.

Cells having different target expression levels were examined—The cells(5×10⁴/well) were sown in a 96-MTP (Greiner bio-one, CELLSTAR, 650 180,U-bottom) in a total volume of 100 μl. After addition of thetarget-specific antibody in a final concentration of 10 μg/ml, thebatches were incubated at 37° C. for different periods of time (1 h, 2h, 6 h, in triplicate). The isotype control was treated under identicalconditions. A parallel batch was treated and incubated constantly at 4°C. (negative control). FACS analysis was carried out using the Guavaflow cytometer (Millipore). Kinetic evaluation was carried out bymeasuring the fluorescence intensity, and evaluation took place usingthe guavaSoft 2.6 software (Millipore). For the targets andtarget-specific antibodies described here, a significant and specificinternalization was detected in various cells; the isotype controlsshowed no internalization.

C-2c Co-Localization Assays of the Anti-CD123 Antibodies

Owing to the linker, the active metabolite of the antibody-drugconjugate is generated by lysosomal degradation. Accordingly,intracellular trafficking after internalization has taken place is ofessential importance. Studies about the co-localization of the antibodyusing labels specific for the lysosomal organelle (e.g. surfacemolecules or small GTPases) allow the selection of antibodies having thedesired profile. To this end, target-positive cells (5×10⁴/well) in atotal volume of 100 μl were sown into a 96-MTP (Greiner bio-one,CELLSTAR, 650 180, U-bottom). Following addition of theCypHer5E-labelled anti-target antibody (final concentation 20 μg/ml),the batches (duplicates per point in time) were incubated at 37° C. for30 min, 2 h and 6 h in an incubator (5% CO₂). 30 min prior to the end ofthe chosen incubation time, the lysosome-specific label was added to thebatches to be examined. The lysosomes were stained with CytoPainterLysoGreen indicator reagent (final concentration 1:2000; abcam,ab176826). After incubation, 200 μl of ice-cold FACS buffer (DULBECCO'SPBS, Sigma-Aldrich, No. D8537+3% FBS heat inactivated FBS, Gibco, No.10500-064) were added and the cell suspension was centrifuged at 400×gand 4° C. for 5 min. The cell pellet was resuspended in 300 μl ice-coldFACS buffer and centrifuged again (4 min, 400×g at 4° C.). Aftercentrifugation, the supernatant was discarded and the cell pellet wastaken up in 30 μl of ice-cold FACS buffer. The samples were thenimmediately subjected to FACS/image analysis (FlowSight amnis,Millipore). Co-localization was evaluated using a special software(co-localization software IDEAS Application v6.1). Table 3 summarizesthe results from this assay in an exemplary manner for anti-CD123antibodies.

TABLE 3 Example Co-localization [%] TPP-9476 29 TPP-8987 28 TPP-8988 41TPP-6013 43 7G3 10 Isotype control 0.2

The humanized antibodies TPP-9476 and TPP-8987 exhibit a markedlyimproved profile compared to the parental murine antibody.

C-3 In Vitro Tests for Determining Cell Permeability

The cell permeability of a substance can be investigated by means of invitro testing in a flux assay using Caco-2 cells [M. D. Troutman and D.R. Thakker, Pharm. Res. 20 (8), 1210-1224 (2003)]. For this purpose, thecells were cultured for 15-16 days on 24-well filter plates. For thedetermination of permeation, the respective test substance was appliedin a HEPES buffer to the cells either apically (A) or basally (B) andincubated for 2 hours. After 0 hours and after 2 hours, samples weretaken from the cis and trans compartments. The samples were separated byHPLC (Agilent 1200, Boblingen, Germany) using reverse phase columns. TheHPLC system was coupled via a Turbo Ion Spray Interface to a TripleQuadropole mass spectrometer API 4000 (AB SCIEX Deutschland GmbH,Darmstadt, Germany). The permeability was evaluated on the basis of aP_(app) value, which was calculated using the formula published bySchwab et al. [D. Schwab et al., J. Med. Chem. 46, 1716-1725 (2003)]. Asubstance was classified as actively transported when the ratio ofP_(app) (B-A) to P_(app) (A-B) (efflux ratio) was >2 or <0.5.

Of critical importance for toxophores which are released intracellularlyis the permeability from B to A [P_(app) (B-A)] and the ratio of P_(app)(B-A) to P_(app) (A-B) (efflux ratio): The lower this permeability, theslower the active and passive transport processes of the substancethrough the monolayer of Caco-2 cells, so that the substance, followingintracellular release, remains in the cell for longer. As a consequenceof the metabolite remaining in the cell for longer, the probability ofinteraction with the biochemical target (here: kinesin spindle proteinKSP/Eg5) is increased, resulting in an improved cytotoxic action.

Table 4 below sets out permeability data for representative workingexamples from this assay:

TABLE 4 Working P_(app) (B-A) Efflux Example [nm/s] ratio M1 2.7 1.6 R3M213 16

The metabolite M1, which can be formed from the binder-drug conjugatesaccording to the invention, exhibits both reduced transport from thecell and a reduced efflux ratio compared with the reference metaboliteR3M, which can be formed from the binder-drug conjugates of ReferenceExamples 2.

C-4 In Vitro Tests for Determining the Substrate Properties forP-Glycoprotein (P-gp)

Many tumour cells express transporter proteins for drugs, and thisfrequently accompanies the development of resistance towardscytostatics. Substances which are not substrates of such transporterproteins, such as P-glycoprotein (P-gp) or BCRP, for example, couldtherefore exhibit an improved activity profile.

The substrate properties of a substance for P-gp (ABCB1) were determinedby means of a flux assay using LLC-PK1 cells which overexpress P-gp(L-MDR1 cells) [A. H. Schinkel et al., J. Clin. Invest. 96, 1698-1705(1995)]. For this purpose, the LLC-PK1 cells or L-MDR1 cells werecultured on 96-well filter plates for 3-4 days. For determination of thepermeation, the respective test substance, alone or in the presence ofan inhibitor (such as ivermectin or verapamil, for example), was appliedin a HEPES buffer to the cells either apically (A) or basally (B) andincubated for 2 hours. After 0 hours and after 2 hours, samples weretaken from the cis and trans compartments. The samples were separated byHPLC using reverse phase columns. The HPLC system was coupled via aTurbo Ion Spray Interface to an API 3000 triple quadropole massspectrometer (Applied Biosystems Applera, Darmstadt, Germany). Thepermeability was evaluated on the basis of a P_(app) value, which wascalculated using the formula published by Schwab et al. [D. Schwab etal., J. Med. Chem. 46, 1716-1725 (2003)]. A substance was classified asP-gp substrate when the efflux ratio of P_(app) (B-A) to P_(app) (A-B)was >2.

As further criteria for the evaluation of the P-gp substrate properties,the efflux ratios in L-MDR1 and LLC-PK1 cells or the efflux ratio in thepresence or absence of an inhibitor may be compared. If these valuesdiffer by a factor of more than 2, the substance in question is a P-gpsubstrate.

C-5 Pharmacokinetics

After i.v. administration of 5 mg/kg of Example 2c-9476 (DAR 6.3) andExample 2c-9476 (DAR 3.4) in male Wistar rats, the plasma concentrationsof the ADCs were measured by ELISA and the pharmacokinetic parameterssuch as clearance (CL), area under the curve (AUC) and half-life(t_(1/2)) were calculated.

Table 5 summarizes the pharmacokinetic parameters of Example 2c-9476with DAR 6.3 and DAR 3.4.

TABLE 5 Example 2c-9476C 2c-9476D DAR 3.4 6.3 Species Rat Rat StrainWistar Wistar Sex male male Administration iv bolus iv bolus Dose admin[mg/kg] 5.0 5.0 AUC_(normal) [kg * h/l] 4897 4069 AUC [mg *h/l] 2448420343 Cl_(matrix) [ml/h/kg] 0.20 0.25 Vss [l/kg] 0.063 0.069 MRT [h] 306281 t_(1/2) [h] 229 219

In this exploratory PK study on rats, for both examples a typical IgGprofile was observed following i.v. administration. No significantdifferences were observed between Example 2c-9476 with DAR 6.3 andExample 2c-9476 with DAR 3.4.

Analysis for Quantification of the ADCs Used

The antibody part of the ADCs was determined using a ligand bindingassay (ELISA) as total IgG concentration in plasma samples and tumourlysates. Here, the sandwich ELISA format was used. This ELISA had beenqualified and validated for the determination in plasma and tumoursamples. The ELISA plates were coated with anti-human goat IgG Fcantibodies. After incubation with the sample, the plates were washed andincubated with a detector conjugate of simian anti-human IgG(H+L)antibody and horseradish peroxidase (HRP). After a further washing step,the HRP substrate was added to OPD and the colour development wasmonitored via absorption at 490 nm. Standard samples having a known IgGconcentration were fitted using a 4-parameter equation. Within the lower(LLOQ) and upper (ULOQ) quantification limits, the unknownconcentrations were determined by interpolation.

C5a: Identification of the ADC Metabolites After Internalization InVitro

Description of the Method:

Internalization studies with immunoconjugates are carried out to analysemetabolites formed intracellularly. To this end, human lung tumour cellsNCI H292 (3×10⁵/well) are sown in 6-well plates and incubated overnight(37° C., 5% CO₂). The cells are treated with 10 μg/ml (66 nM) of the ADCto be examined. Internalization was carried out at 37° C. and 5% CO₂.Cell samples are taken for further analysis at various times (0, 4, 24,48, 72 h). First of all, the supernatants (about 5 ml) are harvestedand, after centrifugation (2 min, RT, 1000 rpm Heraeus Variofuge 3.0R),stored at −80° C. The cells are washed with PBS and detached withAccutase, and the cell number is determined. After another washing, adefined number of cells (2×10⁵) is treated with 100 ml of lysis buffer(Mammalian Cell Lysis Kit (Sigma MCL1) and incubated with continuousshaking (Thermomixer, 15 min, 4° C., 650 rpm) in Protein LoBind tubes(Eppendorf Cat. No. 0030 108.116). After the incubation, the lysate iscentrifuged (10 min, 4° C., 12000 g, eppendorf 5415R) and thesupernatant is harvested. The supernatant obtained is stored at −80° C.All samples are then analysed as follows.

For workup of 50 μl of culture supernatant/cell lysate, 150 μl ofprecipitation reagent (methanol) are added and the mixture is shaken for10 seconds. The precipitation reagent contains an internal standard(ISTD) in a suitable concentration (generally in the range of 20-100μg/l). After centrifugation at 1881 g for 10 minutes, the supernatant istransferred into an autosampler vial, made up with 300 μl of a buffermatched to the eluent and shaken again and centrifuged at 1881 g for 10min.

The cell lysate and supernatant samples are finally analysed using theHPLC-coupled AP16500 triple-quadrupole mass spectrometer from AB SCIEXDeutschland GmbH.

For calibration, blank lysate or blank supernatant is admixed withappropriate concentrations (0.1-1000 μg/l). The detection limit (LLOQ)is about 0.2 μg/l.

Quality controls for testing validity contain 4 and 40 μg/l.

C5b: Identification of the ADC Metabolites In Vivo

After i.v. administration of 3-30 mg/kg of different ADCs, the plasmaand tumour concentrations of the ADCs and any metabolites occuring canbe measured, and the pharmacokinetic parameters such as clearance (CL),area under the curve (AUC) and half-times (t_(1/2)) can be calculated.

Analysis for Quantification of the Potential Metabolites

The analysis of the compounds in the plasma, tumour, liver and kidneyfollows after precipitation of the proteins with generally methanol byhigh-pressure liquid chromatography (HPLC) coupled to atriple-quadrupole mass spectrometer (MS).

For workup of 50 μl of plasma, 150 μl of precipitation reagent(generally methanol) are added and the mixture is shaken for 10 sec. Theprecipitation reagent contains an internal standard (ISTD) in a suitableconcentration (generally in the range of 20-100 μg/l). Aftercentrifugation at 1881 g for 10 minutes, the supernatant is transferredinto an autosampler vial, made up with 300 μl of a buffer matched to theeluent and shaken again.

In the workup of tumour or organ material, the particular material isadmixed with 3-20 times the amount of extraction buffer. The extractionbuffer contains 50 ml of Tissue Protein Extraction Reagent (Pierce,Rockford, Ill.), two pellets of Complete-Protease-Inhibitor-Cocktail(Roche Diagnostics GmbH, Mannheim, Germany) and phenylmethylsulfonylfluoride (Sigma, St. Louis, Mo.) in a final concentration of 1 mM.According to the tissue type (hard: tumour; soft: liver, kidney), thelysis and homogenization programme of the Prescellys 24 lysis andhomogenization system (Bertin Technologies) is selected(www.prescellys.com). The homogenized samples are left to stand at 4° C.overnight. 50 μl of the homogenizate are transferred into an autosamplervial and made up with 150 μl of methanol including ISTD, agitated for 10sec and then left to stand for 5 min. After adding 300 μl of ammoniumacetate buffer (pH 6.8) and agitating briefly, the sample is centrifugedat 1881 g for 10 minutes.

For calibration, plasma for plasma samples and corresponding blankmatrix for tissue samples is admixed with concentrations of 0.6-1000μg/l. According to the sample type or tissue type, the detection limit(LOQ) is between 1 and 20 μg/l.

The plasma and matrix samples are finally analysed using theHPLC-coupled AP14500 triple-quadrupole mass spectrometer from AB SCIEXDeutschland GmbH.

Quality controls for testing validity contain 4, 40 and 400 μg/l.

Table 6 shows metabolite concentrations in the MOLM-13 xenograft mousemodel measured in -tumour, -liver, -kidney and plasma 24 h afteradministration of 5 mg/kg of the ADC from Example 2c-9476 (n=3). Themetabolite measured was: metabolite M1. n.c.=not calculated; LOQ: limitof quantification

TABLE 6 Metabolite M1 Metabolite M1 LOQ MW (μg/l) SD (μg/l) (μg/l)Tumour Example 2c-9476B 59.5 0.3 2.0-20.0 Liver Example 2c-9476B <LOQn.c. 2.5 Kidney Example 2c-9476B 10.9 6.5 5.0 Plasma Example 2c-9476B<LOQ n.c. 1.0

Administration of the ADC Example 2c-9476 according to the inventionhaving a legumain-cleavable linker lead to a markedly selectiveenrichment of the active compound at the target tissue (tumour) comparedto other, healthy organs/tissues.

C-6 Activity Test In Vivo

The activity of the conjugates according to the invention was tested invivo, for example, using xenograft models. The person skilled in the artis familiar with methods in the prior art which allow the activity ofthe compounds according to the invention to be tested (see, for example,WO 2005/081711; Polson et al., Cancer Res. 2009 Mar. 15; 69(6):2358-64).To this end, a tumour cell line expressing the target molecule of thebinder was inoculated into rodents (for example mice). A conjugateaccording to the invention, an isotype antibody control conjugate, acontrol antibody or isotonic saline was then administered to theinoculated animals. The administration took place once or more thanonce. Following an incubation time of several days, the size of thetumour was determined by comparing conjugate-treated animals and thecontrol group. The conjugate-treated animals displayed a smaller tumoursize.

C-6a. Growth Inhibition/Regression of Experimental Tumours in the Mouse

Human tumour cells which express the antigen for the antibody-drugconjugate are inoculated subcutaneously into the flank ofimmunosuppressed mice, for example NMRi nude or SCID mice. 1-10 millioncells are detached from the cell culture, centrifuged and resuspended inmedium or medium/matrigel. The cell suspension is injected under theskin of the mouse.

Within a few days, a tumour grows. Treatment is commenced after thetumour is established, at a tumour size of approximately 40 mm². Toexamine the effect on larger tumours, treatment may be initiated only ata tumour size of 50-100 mm².

Treatment with APDCs and ADCs is carried out via the intravenous (i.v.)route into the tail vein of the mouse. The ADC is administered in avolume of 5 ml/kg.

The treatment protocol depends on the pharmacokinetics of the antibody.With the conjugates according to the invention, treatment is effectedonce per week for 2 or 3 weeks as the standard. For a quick assessment,a protocol with a single treatment may also be suitable. However, thetreatment may also be continued, or a second cycle of three treatmentdays may follow at a later time.

As standard, 8 animals are used per treatment group. In addition to thegroups to which the active substances are administered, one group istreated as control group only with the buffer, according to the sameprotocol.

During the experiment, the tumour area is measured regularly in twodimensions (length/width) using a caliper. The tumour area is determinedas length×width. The ratio of the mean tumour area of the treatmentgroup to that of the control group is stated as T/C area.

When, after the end of the treatment, all groups of the experiment areterminated at the same time, the tumours can be removed and weighed. Theratio of the mean tumour weights of the treatment group to that of thecontrol group is stated as T/C weight.

C-6b. Efficacy of the ADCs According to the Invention in Various TumourModels

The tumour cells (e.g. NCI-H292, REC-1, MOLM-13 and MV-4-11 areinoculated subcutaneously into the flank of female NMRI-nude mice(Janvier). At a tumour size of 40 mm², intravenous treatment is effectedwith the antibody-drug coniugate. After the treatment, monitoring of thetumour growth continues if appropriate.

The treatment with the ADCs according to the invention leads to adistinct and in some cases long-lasting inhibition of tumour growthcompared to the control group and the conjugated isotype controlantibody. Table 7 shows the T/C values determined for tumour area on therespective day of the end of the experiment, calculated from the startof treatment.

TABLE 7 Tumour Dose T/C Example Antigen model Dose scheme area 2k-7007TWEAKR NCI-H292  5 mg/kg Q7dx3 0.09 (human lung (day 25) carcinoma)2x-9574 CXCR5 REC-1 (human 10 mg/kg Q7dx3 0.20 mantle cell (day 24)lymphoma) 2c-9476D CD123 MOLM-13  5 mg/kg Q7dx2 0.15 (human acute (day17) myeloid leukaemia) 2c-9476D CD123 MV-4-11  5 mg/kg Q7dx2 0.16 (human(day 18) acute myeloid leukaemia)

1. A binder-drug conjugate of formula (I)

wherein X₁ is N, X₂ is N and X₃ is C; or X₁ is N, X₂ is C and X₃ is N;or X₁ is CH or CF, X₂ is C and X₃ is N; or X₁ is NH, X₂ is C and X₃ isC; or X₁ is CH, X₂ is N and X₃ is C; R¹ is hydrogen or methyl; R² ismethyl, ethyl, —CH₂—CH(CH₃)₂, —CH₂—C(═O)OH or isopropyl; R³ is methyl,ethyl, —CH₂—CH(CH₃)₂ or —CH₂—C(═O)—NH₂; M is the group#-C(═O)—CH(CH₃)—NH—C(═O)—CH₂—NH—C(═O)—CH₂—CH(##)-COOH,#-C(═O)—CH(CH₃)—NH—C(═O)—CH₂—NH—C(═O)—CH(##)-CH₂—COOH,#-C(═O)—CH(CH₃)—NH—C(═O)—CH₂—, #-C(═O)—CH₂—NH—C(═O)—CH₂—CH(##)-COOH,#-C(═O)—CH₂—NH—C(═O)—CH(##)-CH₂—COOH, #-C(═O)—CH₂—W,#-C(═O)—CH(CH₃)—NH—C(═O)—(CH₂)₂₋₈—C(═O)-###, #-C(═O)—(CH₂)₃—C(═O)-###,#-C(═O)—CH(CH₃)—NH—C(═O)—(CH₂)₅—W, #-C(═O)—CH(CH₃)—NH—C(═O)—(CH₂)-##, or#-C(═O)—CH(CH₃)—NH—C(═O)—(CH₂—CH₂—O)₁₋₈—(CH₂)₂—NH—C(═O)—CH₂-##; W is thegroup

n is a number from 1 to 50; AK is a binder or a derivative thereof; #represents the bond to the compound; ## represents the bond to a sulfuratom of a cysteine side-chain of the binder; and ### represents the bondto a nitrogen atom of a lysine side-chain of the binder, or a salt, asolvate, or a salt of the solvate thereof.
 2. The binder-drug conjugateaccording to claim 1, wherein: X₁ is CH, X₂ is C, X₃ is N; R¹ ishydrogen or methyl; R² is methyl, —CH₂—CH(CH₃)₂, —CH₂—C(═O)OH orisopropyl; R³ is methyl, —CH₂—CH(CH₃)₂ or —CH₂—C(═O)—NH₂; M is the group#-C(═O)—CH(CH₃)—NH—C(═O)—CH₂—NH—C(═O)—CH₂—CH(##)-COOH,#-C(═O)—CH(CH₃)—NH—C(═O)—CH₂—NH—C(═O)—CH(##)-CH₂—COOH,#-C(═O)—CH(CH₃)—NH—C(═O)—CH₂—W, #-C(═O)—CH₂—NH—C(═O)—CH₂—CH(##)-COOH,#-C(═O)—CH₂—NH—C(═O)—CH(##)-CH₂—COOH, #-C(═O)—CH₂—W,#-C(═O)—CH(CH₃)—NH—C(═O)—(CH₂)₃—C(═O)-###, #-C(═O)—(CH₂)₃—C(═O)-###,#-C(═O)—CH(CH₃)—NH—C(═O)—(CH₂)₅—W, #-C(═O)—CH(CH₃)—NH—C(═O)—(CH₂)-##, or#-C(═O)—CH(CH₃)—NH—C(═O)—(CH₂—CH₂—O)₄—(CH₂)₂—NH—C(═O)—CH₂-##; W is thegroup

n is a number from 1 to 50; AK is a binder or a derivative thereof; #represents the bond to the compound; ## represents the bond to a sulfuratom of a cysteine side-chain of the binder; and ### represents the bondto a nitrogen atom of a lysine side-chain of the binder, or a salt, asolvate, or a salt of the solvate thereof.
 3. The binder-drug conjugateaccording to claim 1, wherein: R¹ is hydrogen or methyl; R² is methyl orisopropyl; R³ is methyl or —CH₂—C(═O)—NH₂; M is the group#-C(═O)—CH(CH₃)—NH—C(═O)—(CH₂)₃—C(═O)-###; n is a number from 1 to 50;AK is a binder or a derivative thereof; # represents the bond to thecompound; ### represents the bond to a nitrogen atom of a lysineside-chain of the binder, or a salt, a solvate, or a salt of the solvatethereof.
 4. The binder-drug conjugate according to claim 1, wherein: R¹is methyl; R² is methyl; R³ is —CH₂—C(═O)—NH₂; M is the group#-C(═O)—CH(CH₃)—NH—C(═O)—(CH₂)₃—C(═O)-###; n is a number from 1 to 50;AK is a binder or a derivative thereof; # represents the bond to thecompound; and ### represents the bond to a nitrogen atom of a lysineside-chain of the binder, or a salt, a solvate, or a salt of the solvatethereof.
 5. The binder-drug conjugate according to claim 1, wherein: R¹is methyl; R² is methyl; R³ is —CH₂—C(═O)—NH₂; M is the group#-C(═O)—CH(CH₃)—NH—C(═O)—(CH₂)₃—C(═O)-###; n is a number from 1 to 20;AK is a binder or a derivative thereof; # represents the bond to thecompound; and ### represents the bond to a nitrogen atom of a lysineside-chain of the binder, or a salt, a solvate, or a salt of the solvatethereof.
 6. The binder-drug conjugate according to claim 1, wherein: R¹is methyl; R² is methyl; R³ is —CH₂—C(═O)—NH₂; M is the group#-C(═O)—CH(CH₃)—NH—C(═O)—(CH₂)₃—C(═O)-###; n is a number from 1 to 20;AK is an anti-CD123 antibody, an anti-CXCR5 antibody, an anti-B7H3antibody, an anti-TWEAKR antibody, an anti-Her2 antibody or an anti-EGFRantibody or is an antigen-binding antibody fragment of these; #represents the bond to the compound; and ### represents the bond to anitrogen atom of a lysine side-chain of the antibody (AK) or of theantigen-binding antibody fragment thereof, or a salt, a solvate, or asalt of the solvate thereof.
 7. The binder-drug conjugate according toclaim 1, wherein: R¹ is methyl; R² is methyl; R³ is —CH₂—C(═O)—NH₂; M isthe group #-C(═O)—CH(CH₃)—NH—C(═O)—(CH₂)₃—C(═O)-###; n is a number from1 to 20; AK is an anti-CD123 antibody selected from the group consistingof TPP-9476, TPP-8988, TPP-8987 and TPP-6013, or is an anti-CXCR5antibody selected from the group consisting of TPP-9574 and TPP-9580, oris an anti-B7H3 antibody TPP-8382, or is an anti-TWEAKR antibodyselected from the group consisting of TPP-7006 and TPP-7007, or is ananti-Her2 antibody TPP-1015, or is an anti-EGFR antibody TPP-981, or isan antigen-binding antibody fragment of these; # represents the bond tothe compound; ### represents the bond to a nitrogen atom of a lysineside-chain of the antibody (AK) or of the antigen-binding antibodyfragment thereof, or a salt, a solvate, or a salt of the solvatethereof.
 8. The binder-drug conjugate according to claim 1, wherein thebinder-drug conjugate has a structure selected from the group consistingof:

wherein AK1 is an antibody which is attached via a sulfur atom of acysteine side-chain, AK2 is an antibody which is attached via a nitrogenatom of a lysine side-chain, and N is a number from 1 to 50, or a salt,a solvate, or a salt of the solvate thereof.
 9. The binder-drugconjugate according to claim 8, wherein: n is a number from 1 to 20, ora salt, a solvate, or a salt of the solvate thereof.
 10. The binder-drugconjugate according to claim 9, wherein: n is a number from 1 to 8, or asalt, a solvate, or a salt of the solvate thereof.
 11. The binder-drugconjugate according to claim 10, wherein: n is a number from 4 to 8, ora salt, a solvate, or a salt of the solvate thereof.
 12. The binder-drugconjugate according to claim 1, wherein: AK is an antibody selected fromthe group consisting of TPP-8382 (anti B7H3), TPP-6013 (anti-CD123),TPP-8987 (anti-CD123), TPP-8988 (anti-CD123), TPP 9476 (anti-CD123),TPP-9580 (anti-CXCR5) and TPP 9574 (anti-CXCR5), or an antigen-bindingfragment of these.
 13. The binder-drug conjugate according to claim 1,wherein: AK is an anti-CD123 antibody TPP-6013, is an anti-CD123antibody TPP-8987, is an anti-CD123 antibody TPP-8988 or is ananti-CD123 antibody TPP-9476, or an antigen-binding fragment of these.14. The binder-drug conjugate according to claim 1, wherein AK (i) is ananti-B7H3 antibody comprising a variable region of the heavy chain (VH)comprising the variable CDR1 sequence of the heavy chain (H-CDR1), asshown by SEQ ID NO: 52, the variable CDR2 sequence of the heavy chain(H-CDR2), as shown by SEQ ID NO: 53 and the variable CDR3 sequence ofthe heavy chain (H-CDR3), as shown by SEQ ID NO: 54, and a variableregion of the light chain (VL) comprising the variable CDR1 sequence ofthe light chain (L-CDR1), as shown by SEQ ID NO: 56, the variable CDR2sequence of the light chain (L-CDR2), as shown by SEQ ID NO: 57 and thevariable CDR3 sequence of the light chain (L-CDR3), as shown by SEQ IDNO: 58, (ii) is an anti-CD123 antibody comprising a variable region ofthe heavy chain (VH) comprising the variable CDR1 sequence of the heavychain (H-CDR1), as shown by SEQ ID NO: 22, the variable CDR2 sequence ofthe heavy chain (H-CDR2), as shown by SEQ ID NO: 23 and the variableCDR3 sequence of the heavy chain (H-CDR3), as shown by SEQ ID NO: 24,and a variable region of the light chain (VL) comprising the variableCDR1 sequence of the light chain (L-CDR1), as shown by SEQ ID NO: 26,the variable CDR2 sequence of the light chain (L-CDR2), as shown by SEQID NO: 27 and the variable CDR3 sequence of the light chain (L-CDR3), asshown by SEQ ID NO: 28, (iii) is an anti-CD123 antibody comprising avariable region of the heavy chain (VH) comprising the variable CDR1sequence of the heavy chain (H-CDR1), as shown by SEQ ID NO: 62, thevariable CDR2 sequence of the heavy chain (H-CDR2), as shown by SEQ IDNO: 63 and the variable CDR3 sequence of the heavy chain (H-CDR3), asshown by SEQ ID NO: 64, and a variable region of the light chain (VL)comprising the variable CDR1 sequence of the light chain (L-CDR1), asshown by SEQ ID NO: 66, the variable CDR2 sequence of the light chain(L-CDR2), as shown by SEQ ID NO: 67 and the variable CDR3 sequence ofthe light chain (L-CDR3), as shown by SEQ ID NO: 68, (iv) is ananti-CD123 antibody comprising a variable region of the heavy chain (VH)comprising the variable CDR1 sequence of the heavy chain (H-CDR1), asshown by SEQ ID NO: 72, the variable CDR2 sequence of the heavy chain(H-CDR2), as shown by SEQ ID NO: 73 and the variable CDR3 sequence ofthe heavy chain (H-CDR3), as shown by SEQ ID NO: 74, and a variableregion of the light chain (VL) comprising the variable CDR1 sequence ofthe light chain (L-CDR1), as shown by SEQ ID NO: 76, the variable CDR2sequence of the light chain (L-CDR2), as shown by SEQ ID NO: 77 and thevariable CDR3 sequence of the light chain (L-CDR3), as shown by SEQ IDNO: 78, (v) is an anti-CD123 antibody comprising a variable region ofthe heavy chain (VH) comprising the variable CDR1 sequence of the heavychain (H-CDR1), as shown by SEQ ID NO: 82, the variable CDR2 sequence ofthe heavy chain (H-CDR2), as shown by SEQ ID NO: 83 and the variableCDR3 sequence of the heavy chain (H-CDR3), as shown by SEQ ID NO: 84,and a variable region of the light chain (VL) comprising the variableCDR1 sequence of the light chain (L-CDR1), as shown by SEQ ID NO: 86,the variable CDR2 sequence of the light chain (L-CDR2), as shown by SEQID NO: 87 and the variable CDR3 sequence of the light chain (L-CDR3), asshown by SEQ ID NO: 88, (vi) is an anti-CXCR5 antibody comprising avariable region of the heavy chain (VH) comprising the variable CDR1sequence of the heavy chain (H-CDR1), as shown by SEQ ID NO: 92, thevariable CDR2 sequence of the heavy chain (H-CDR2), as shown by SEQ IDNO: 93 and the variable CDR3 sequence of the heavy chain (H-CDR3), asshown by SEQ ID NO: 94, and a variable region of the light chain (VL)comprising the variable CDR1 sequence of the light chain (L-CDR1), asshown by SEQ ID NO: 96, the variable CDR2 sequence of the light chain(L-CDR2), as shown by SEQ ID NO: 97 and the variable CDR3 sequence ofthe light chain (L-CDR3), as shown by SEQ ID NO: 98, or (vii) is ananti-CXCR5 antibody comprising a variable region of the heavy chain (VH)comprising the variable CDR1 sequence of the heavy chain (H-CDR1), asshown by SEQ ID NO: 102, the variable CDR2 sequence of the heavy chain(H-CDR2), as shown by SEQ ID NO: 103 and the variable CDR3 sequence ofthe heavy chain (H-CDR3), as shown by SEQ ID NO: 104, and a variableregion of the light chain (VL) comprising the variable CDR1 sequence ofthe light chain (L-CDR1), as shown by SEQ ID NO: 106, the variable CDR2sequence of the light chain (L-CDR2), as shown by SEQ ID NO: 107 and thevariable CDR3 sequence of the light chain (L-CDR3), as shown by SEQ IDNO: 108, or is an antigen-binding fragment of these antibodies.
 15. Thebinder-drug conjugate according to claim 1, wherein AK (i) is ananti-B7H3 antibody comprising a variable region of the heavy chain (VH)as shown in SEQ ID NO: 51 and a variable region of the light chain (VL)as shown in SEQ ID NO: 55, (ii) is an anti-CD123 antibody comprising avariable region of the heavy chain (VH) as shown in SEQ ID NO: 21 and avariable region of the light chain (VL) as shown in SEQ ID NO: 25, (iii)is an anti-CD123 antibody comprising a variable region of the heavychain (VH) as shown in SEQ ID NO: 61 and a variable region of the lightchain (VL) as shown in SEQ ID NO: 65, (iv) is an anti-CD123 antibodycomprising a variable region of the heavy chain (VH) as shown in SEQ IDNO: 71 and a variable region of the light chain (VL) as shown in SEQ IDNO: 75, (v) is an anti-CD123 antibody comprising a variable region ofthe heavy chain (VH) as shown in SEQ ID NO: 81 and a variable region ofthe light chain (VL) as shown in SEQ ID NO: 85, (vi) is an anti-CXCR5antibody comprising a variable region of the heavy chain (VH) as shownin SEQ ID NO: 91 and a variable region of the light chain (VL) as shownin SEQ ID NO: 95, (vii) is an anti-CXCR5 antibody comprising a variableregion of the heavy chain (VH) as shown in SEQ ID NO: 101 and a variableregion of the light chain (VL) as shown in SEQ ID NO: 105, or is anantigen-binding fragment of these antibodies.
 16. The binder-drugconjugate according to claim 1, wherein AK (i) is an anti-B7H3 antibodycomprising a region of the heavy chain as shown in SEQ ID NO: 59 and aregion of the light chain as shown in SEQ ID NO: 60, (ii) represents isan anti-CD123 antibody comprising a region of the heavy chain as shownin SEQ ID NO: 29 and a region of the light chain as shown in SEQ ID NO:30, (iii) is an anti-CD123 antibody comprising a region of the heavychain as shown in SEQ ID NO: 69 and a region of the light chain as shownin SEQ ID NO: 70, (iv) is an anti-CD123 antibody comprising a region ofthe heavy chain as shown in SEQ ID NO: 79 and a region of the lightchain as shown in SEQ ID NO: 80, (v) is an anti-CD123 antibodycomprising a region of the heavy chain as shown in SEQ ID NO: 89 and aregion of the light chain as shown in SEQ ID NO: 90, (vi) is ananti-CXCR5 antibody comprising a region of the heavy chain as shown inSEQ ID NO: 99 and a region of the light chain as shown in SEQ ID NO:100, or (vii) is an anti-CXCR5 antibody comprising a region of the heavychain as shown in SEQ ID NO: 109 and a region of the light chain asshown in SEQ ID NO: 110, or is an antigen-binding fragment of theseantibodies.
 17. The binder-drug conjugate according to claim 1, whereinAK is an antibody or an antigen-binding antibody fragment thereof, andwherein the antibody or the antigen-binding antibody fragment binds toan extracellular target molecule.
 18. The binder-drug conjugateaccording to claim 17, wherein the antibody or the antigen-bindingantibody fragment binds to an extracellular cancer target molecule. 19.The binder-drug conjugate according to claim 18, wherein the antibody orthe antigen-binding antibody fragment, after binding to itsextracellular target molecule on the target cell, is internalized by thetarget cell through the binding.
 20. A pharmaceutical compositioncomprising at least one binder-drug conjugate according to claim 1 incombination with an inert, non-toxic, pharmaceutically suitableauxiliary.
 21. (canceled)
 22. A method for treatment ofhyperproliferative or angiogenic disorders, comprising administering toa human in need thereof an effective amount of the binder-drug conjugateaccording to claim
 1. 23. A method for treatment of cancer and tumours,comprising administering to a human in need thereof an effective amountof the binder-drug conjugate according to claim
 1. 24. A method fortreatment of cancer and tumours, comprising administering to a human inneed thereof an effective amount of the binder-drug conjugate accordingto claim 1 in combination with one or more therapeutic approaches forcancer immunotherapy or with one or more active compounds directedagainst a molecular target of cancer immunotherapy.